Bismuth-thiols as antiseptics for biomedical uses, including treatment of bacterial biofilms and other uses

ABSTRACT

Compositions and methods, including novel homogeneous microparticulate suspensions, are described for treating natural surfaces that contain bacterial biofilm, including unexpected synergy or enhancing effects between bismuth-thiol (BT) compounds and certain antibiotics, to provide formulations including antiseptic formulations. Previously unpredicted antibacterial properties and anti-biofilm properties of disclosed BT compounds and BT compound-plus-antibiotic combinations are also described, including preferential efficacies of certain such compositions for treating certain gram-positive bacterial infections, and distinct preferential efficacies of certain such compositions for treating certain gram-negative bacterial infections.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US2011/023549 filed Feb. 3, 2011; which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application No. 61/373,188 filed Aug.12, 2010; and which also claims the benefit under 35 U.S.C. §120 of PCTApplication No. PCT/US2010/023108, filed Feb. 3, 2010; each of whichprior applications is incorporated herein by reference in its entirety.This application is also a continuation-in-part of PCT Application No.PCT/US2010/023108, filed Feb. 3, 2010; which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application No. 61/149,593, filedFeb. 3, 2009; and this application is also a continuation-in-part ofU.S. patent application Ser. No. 12/699,680, filed Feb. 3, 2010; whichclaims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 61/149,593, filed Feb. 3, 2009; each of which priorapplications is incorporated herein by reference in its entirety.

BACKGROUND

1. Technical Field

The presently disclosed invention embodiments relate to compositions andmethods for the treatment of microbial infections. In particular, thepresent embodiments relate to improved treatments for managing bacterialinfections in epithelial tissues, including in wounds such as chronicwounds and acute wounds, and in clinical, personal healthcare, and othercontexts, including treatment of bacterial biofilms and otherconditions.

2. Description of the Related Art

The complex series of coordinated cellular and molecular interactionsthat contribute to skin wound healing and responding to and resistingmicrobial infections and/or to healing or maintenance of bodily tissuesgenerally, may be adversely impacted by a variety of external factors,such as opportunistic and nosocomial infections (e.g., clinical regimensthat can increase the risk of infection), local or systemicadministration of antibiotics (which may influence cell growth,migration or other functions and can also select forantibiotic-resistant microbes), frequent wound dressing changes,open-air exposure of wounds to speed healing, the use of temporaryartificial structural support matrix or scaffold materials, the possibleneed for debridement and/or repeat surgery to excise infected ornecrotic tissue and/or other factors.

Wound healing thus continues to be a formidable challenge for clinicalpractitioners worldwide. The current treatments for recalcitrant woundsare impractical and ineffective, often requiring multiple surgeries toclose the wound. For instance, becaplermin, sold under the trademarkREGRANEX® (Ortho-McNeil Pharmaceutical, Inc., available from Ethicon,Inc., recombinant platelet-derived growth factor) exemplifies one of thefew available treatments for chronic wounds, but is expensive to produceand has limited clinical utility.

Chronic and Acute Wounds and Wound Biofilms

Wounds occur when the continuity between cells within a tissue, orbetween tissues, is disrupted, for instance, by physical, mechanical,biological, pathological and/or chemical forces (e.g., burns, dermalinfections, puncture wounds, gunshot or shrapnel wounds, skin ulcers,radiation poisoning, malignancies, gangrene, autoimmune disease,immunodeficiency disease, respiratory insult such as by inhalation orinfection, gastrointestinal insult such as by deleterious ingestion orinfection, circulatory and hematologic disorders including clottingdefects,) or other traumatic injuries, or the like.

While a limited level of bacterial contamination in a wound, or“colonization” of the wound, may not necessarily interfere with theprocesses of wound healing, the presence of bacteria in numberssufficient to overwhelm the host immune defenses can lead to an acutewound or a chronic wound or a wound in which a bacterial biofilm ispresent, such as a wound infection in which bacterial growth proceeds tothe detriment of the host. Bryant and Nix, Acute and Chronic Wounds:Current Management Concepts, 2006 Mosby (Elsevier), NY; Baronoski, WoundCare Essentials: Practical Principles (2^(nd) Ed.), 2007 Lippincott,Williams and Wilkins, Philadelphia, Pa.). For example, acute wounds suchas may result from injury, trauma, surgical intervention, or othercauses, typically lack underlying health deficits and heal rapidly, butmay on occasion fail to do so due to the presence of an infection;rapidly forming bacterial biofilms have been described in acute wounds(e.g., WO/2007/061942). Additional factors that may contribute to thedevelopment of chronic wounds include losses in mobility (e.g., thatresult in continued pressure being applied to a wound site), deficits ofsensation or mental ability, inaccessibility of the wound site (e.g., inthe respiratory or gastrointestinal tracts) and circulatory deficits.Infection at a chronic wound site may be detected by the clinical signsof skin redness, edema, pus formation and/or unpleasant odor, or otherrelevant, clinically accepted criteria.

Acute wounds that cannot heal properly may thus be present, and chronicwounds thus may develop, in higher organisms (including but not limitedto humans and other mammals) when the host's immune system has beenoverwhelmed by bacterial infection of a wound site (e.g., an acutewound), creating permissive conditions for bacteria to invade andfurther destroy tissue. In general, chronic wounds are wounds that donot heal within three months, and instead of becoming smaller they tendto grow larger as the bacterial infiltration progresses. Chronic woundsmay become very painful and stressful for the patient when nearby nervesbecome damaged (neuropathy) as the wound progresses. These wounds affectfour million Americans each year and cost about $9 billion in treatmentexpenses. Afflicted individuals are mostly over the age of 60.

Chronic wounds may in some cases originate as acute wounds and thus mayinclude, for example, gunshot or shrapnel wounds, burns, punctures,venous ulcers, pressure ulcers, diabetic ulcers, radiation poisoning,malignancies, dermal infections, gangrene, surgical wounds, diabeticfoot ulcers, dicubitis ulcers, venous leg ulcers, infected and/orbiofilm-containing nonhealing surgical wounds, pyoderma gangrenosum,traumatic wounds, acute arterial insufficiency, necrotizing fasciitis,osteomyelitis (bone infection), and radiation injuries, such asosteoradionecrosis and soft tissue radionecrosis, or other types ofwounds. Venous ulcers, for example, occur mostly in the legs, as aresult of poor circulation (e.g., ischemia), malfunctioning valves ofveins, or repeated physical trauma (e.g., repetitive injury). Pressureulcers may be present when local pressure that is exerted at or around awound site is greater than blood pressure, for instance, such that poorcirculation, paralysis, and/or bed sores may contribute to, orexacerbate, the chronic wound. Diabetic ulcers may occur in individualswith diabetes mellitus, for example, persons in whom uncontrolled highblood sugar can contribute to a loss of feeling in the extremities,leading to repetitive injuries and/or neglect on the part of theindividual to attend to injuries. Factors that can complicate orotherwise influence clinical onset and outcome of chronic wounds includethe subject's immunological status (e.g., immune suppression,pathologically (e.g., HIV-AIDS), radiotherapeutically orpharmacologically compromised immune system; age; stress); skin aging(including photochemical aging), and development and progression ofbiofilms within the wound. In the case of epithelial tissues in therespiratory and/or gastrointestinal tracts, inaccessibility, occlusion,difficulty in generating epithelial surface-clearing fluid forces ordevelopment of localized microenvironments conducive to microbialsurvival can engender clinical complications.

Wound-related injuries may be accompanied by lost or compromised organfunction, shock, bleeding and/or thrombosis, cell death (e.g., necrosisand/or apoptosis), stress and/or microbial infection. Any or all ofthese events, and especially infection, can delay or prevent theeffective tissue repair processes that are involved in wound healing.Hence, it can be important as early as possible in an individual who hassustained a wound to remove nonviable tissue from a wound site, aprocess referred to as debridement, and also to remove any foreignmatter from the wound site, also referred to as wound cleansing.

Severe wounds, acute wounds, chronic wounds, burns, and ulcers canbenefit from cellular wound dressings. Several artificial skin productsare available for nonhealing wounds or burns such as the artificial skinproducts sold under the trademarks: APLIGRAFT® (Norvartis), DERMAGRAFT®,BIOBRANE®, TRANSCYTE® (Advance Tissue Science), INTEGRA® DERMALREGENERATION TEMPLATE® (from Integra Life Sciences Technology), andORCEL®. These products, however, are not designed to address the problemof bacterial tissue infiltration and wound spreading.

Unfortunately, systemic antibiotics are not effective for the treatmentof chronic wounds, and are generally not used unless an acute bacterialinfection is present. Current approaches include administration orapplication of antibiotics, but such remedies may promote the advent ofantibiotic-resistant bacterial strains and/or may be ineffective againstbacterial biofilms. It therefore may become especially important to useantiseptics when drug resistant bacteria (e.g., methicillin resistantStaphylococcus aureus, or MRSA) are detected. There are many antisepticswidely in use, but bacterial populations or subpopulations that areestablished may not respond to these agents, or to any other currentlyavailable treatments. Additionally, a number of antiseptics may be toxicto host cells at the concentrations that may be needed to be effectiveagainst an established bacterial infection, and hence such antisepticsare unsuitable. This problem may be particularly acute in the case ofefforts to clear infections from natural surfaces, including internalepithelial surfaces, such as respiratory (e.g., airway, nasopharyngealand laryngeal paths, tracheal, pulmonary, bronchi, bronchioles, alveoli,etc.) or gastrointestinal (e.g., buccal, esophageal, gastric,intestinal, rectal, anal, etc.) tracts, or other epithelial surfaces.

Particularly problematic are infections composed of bacterial biofilms,a relatively recently recognized organization of bacteria by which free,single-celled (“planktonic”) bacteria assemble by intercellular adhesioninto organized, multi-cellular communities (biofilms) having markedlydifferent patterns of behavior, gene expression, and susceptibility toenvironmental agents including antibiotics. Biofilms may deploybiological defense mechanisms not found in planktonic bacteria, whichmechanisms can protect the biofilm community against antibiotics andhost immune responses. Established biofilms can arrest thetissue-healing process.

Common microbiologic contaminants that underlie persistent andpotentially deleterious infections include S. aureus, including MRSA(Methicillin Resistant Staphylococcus aureus), Enterococci, E. coli, P.aeruginosa, Streptococci, and Acinetobacter baumannii. Some of theseorganisms exhibit an ability to survive on non-nutritive clinicalsurfaces for months. S. aureus, has been shown to be viable for fourweeks on dry glass, and for between three and six months on dried bloodand cotton fibers (Domenico et al., 1999 Infect. Immun. 67:664-669).Both E. coli and P. aeruginosa have been shown to survive even longerthan S. aureus on dried blood and cotton fibers (ibid).

Microbial biofilms are associated with substantially increasedresistance to both disinfectants and antibiotics. Biofilm morphologyresults when bacteria and/or fungi attach to surfaces. This attachmenttriggers an altered transcription of genes, resulting in the secretionof a remarkably resilient and difficult to penetrate polysaccharidematrix, protecting the microbes. Biofilms are very resistant to themammalian immune system, in addition to their very substantialresistance to antibiotics. Biofilms are very difficult to eradicate oncethey become established, so preventing biofilm formation is a veryimportant clinical priority. Recent research has shown that open woundscan quickly become contaminated by biofilms. These microbial biofilmsare thought to delay wound healing, and are very likely related to theestablishment of serious wound infections.

The current guidelines for the care for military wounds, for example,specify vigorous and complete irrigation and debridement (BlankenshipCL, Guidelines for care of open combat casualty wounds, Fleet Operationsand Support. U.S. Bureau of Medicine and Surgery). While this earlyintervention is important, it is not adequate to prevent the developmentof infection. Additional therapeutic steps need to be taken followingdebridement to promote healing, reduce the microbial bio-burden, andthereby reduce the chances of establishing wound infections and woundbiofilms.

Because of the complex nature of military traumatic wounds, thepotential for infection is great, particularly considering theintroduction of foreign objects and other environmental contaminatingagents. Both military and clinical environments (including people withinboth of these environments) act as important sources of potentiallypathogenic microbes, particularly to those suffering from open and/orcomplex wounds. Acute and chronic wounds, including surgical andmilitary wounds, have already compromised the body's primary defense andbarrier against infection; the skin. Wounds thus expose the interior ofthe body (a moist and nutritive environment) to opportunistic andpathogenic infections. Many of these infections, particularly persistentwound infections, are likely related to biofilm formation, as has beenshown to be the case with chronic wounds (James et al., 2008). Infectionof wounds in hospitals constitutes one of the most common causes ofnosocomial infection, and wounds acquired in military and naturaldisaster environments are particularly susceptible to microbialcontamination. Military wounds are predisposed to infection because theyare typically associated with tissue damage, tend to be extensive anddeep, may introduce foreign bodies and interfere with local bloodsupply, may be associated with fractures and burns, and may lead toshock and compromised immune defenses.

Skin Architecture and Wound Healing

Maintenance of intact, functioning skin and other epithelial tissues(e.g., generally avascular epithelial surfaces that form barriersbetween an organism and its external environment, such as those found inskin and also found in the linings of respiratory and gastrointestinaltracts, glandular tissues, etc.) is significant to the health andsurvival of humans and other animals. The skin is the largest body organin humans and other higher vertebrates (e.g., mammals), protectingagainst environmental insults through its barrier function, mechanicalstrength and imperviousness to water. As a significant environmentalinterface, skin provides a protective body covering that permitsmaintenance of physiological equilibria.

Skin architecture is well known. Briefly, epidermis, the skin outerlayer, is covered by the stratum corneum, a protective layer of deadepidermal skin cells (e.g., keratinocytes) and extracellular connectivetissue proteins. The epidermis undergoes a continual process of beingsloughed off as it is replaced by new material pushed up from theunderlying epidermal granular cell, spinous cell, and basal cell layers,where continuous cell division and protein synthesis produce new skincells and skin proteins (e.g., keratin, collagen). The dermis liesunderneath the epidermis, and is a site for the elaboration by dermalfibroblasts of connective tissue proteins (e.g., collagen, elastin,etc.) that assemble into extracellular matrix and fibrous structuresthat confer flexibility, strength and elasticity to the skin. Alsopresent in the dermis are nerves, blood vessels, smooth muscle cells,hair follicles and sebaceous glands.

As the body's first line of defense, the skin is a major target forclinical insults such as physical, mechanical, chemical and biological(e.g., xenobiotic, autoimmune) attack that can alter its structure andfunction. The skin is also regarded as an important component ofimmunological defense of the organism. In the skin can be foundmigrating as well as resident white blood cells (e.g., lymphocytes,macrophages, mast cells) and epidermal dendritic (Langerhans) cellshaving potent antigen-presenting activity, which contribute toimmunological protection. Pigmented melanocytes in the basal layerabsorb potentially harmful ultraviolet (UV) radiation. Disruption of theskin presents undesirable risks to a subject, including those associatedwith opportunistic infections, incomplete or inappropriate tissueremodeling, scarring, impaired mobility, pain and/or othercomplications. Like the skin, other epithelial surfaces (e.g.,respiratory tract, gastrointestinal tract and glandular linings) havedefined structural attributes when healthy such that infection or otherdisruptions may present serious health risks.

Damaged or broken skin may result, for example, from wounds such ascuts, scrapes, abrasions, punctures, burns (including chemical burns),infections, temperature extremes, incisions (e.g., surgical incisions),trauma and other injuries. Efficient skin repair via wound healing istherefore clearly desirable in these and similar contexts.

Although skin naturally exhibits remarkable ability for self-repairfollowing many types of damage, there remain a number of contexts inwhich skin healing does not occur rapidly enough and/or in whichinappropriate cellular tissue repair mechanisms result in incompletelyremodeled skin that as a consequence can lack the integrity, barrierproperties, mechanical strength, elasticity, flexibility, or otherdesirable properties of undamaged skin. Skin wound healing thus presentssuch associated challenges, for example, in the context of chronicwounds.

Wound healing occurs in three dynamic and overlapping phases, beginningwith the formation of a fibrin clot. The clot provides a temporaryshield and a reservoir of growth factors that attracts cells into thewound. It also serves as a provisional extracellular matrix (ECM) thatthe cells invade during repair. Intermingled with clot formation is theinflammatory phase, which is characterized by the infiltration ofphagocytes and neutrophils into the wound, which clear the wound ofdebris and bacteria, while releasing growth factors that amplify theearly healing response. The process of restoring the denuded area isinitiated in the proliferation phase of healing and is driven bychemokines, cytokines, and proteases that have been secreted from theimmune cells and are concentrated within the clot. Keratinocytes arestimulated to proliferate and migrate, which forms the new layer ofepithelium that covers the wound while wound angiogenesis deliversoxygen, nutrients, and inflammatory cells to the wounded area. Theremodeling phase is the final phase of wound repair and it is carriedout by the myofibroblasts, which facilitate connective tissuecontraction, increase wound strength, and deposit the ECM that forms thescar (Martin, P. Wound Healing-Aiming for Perfect Skin Regeneration.Science 1997; 4:75-80).

Bismuth Thiol-(BT) Based Antiseptics

A number of natural products (e.g., antibiotics) and synthetic chemicalshaving antimicrobial, and in particular antibacterial, properties areknown in the art and have been at least partially characterized bychemical structures and by antimicrobial effects, such as ability tokill microbes (“cidal” effects such as bacteriocidal properties),ability to halt or impair microbial growth (“static” effects such asbacteriostatic properties), or ability to interfere with microbialfunctions such as colonizing or infecting a site, bacterial secretion ofexopolysaccharides and/or conversion from planktonic to biofilmpopulations or expansion of biofilm formation. Antibiotics,disinfectants, antiseptics and the like (including bismuth-thiol or BTcompounds) are discussed, for example, in U.S. Pat. No. 6,582,719,including factors that influence the selection and use of suchcompositions, including, e.g., bacteriocidal or bacteriostaticpotencies, effective concentrations, and risks of toxicity to hosttissues.

Bismuth, a group V metal, is an element that (like silver) possessesantimicrobial properties. Bismuth by itself may not be therapeuticallyuseful and may exhibit certain inappropriate properties, and so mayinstead be typically administered by means of delivery with a complexingagent, carrier, and/or other vehicle, the most common example of whichis, the treatment preparation for gastro-intestinal disorders sold underthe trademark PEPTO-BISMOL® (Procter & Gamble), in which bismuth iscombined (chelated) with subsalicylate. Previous research has determinedthat the combination of certain thiol-(-SH, sulfhydryl) containingcompounds such as ethane dithiol with bismuth, to provide an exemplarybismuth thiol (BT) compound, improves the antimicrobial potency ofbismuth, compared to other bismuth preparations currently available.There are many thiol compounds that may be used to produce BTs(disclosed, for example, in Domenico et al., 2001 Antimicrob. Agent.Chemotherap. 45(5):1417-1421, Domenico et al., 1997 Antimicrob. Agent.Chemother. 41(8):1697-1703, and in U.S. Pat. No. RE37,793, U.S. Pat. No.6,248,371, U.S. Pat. No. 6,086,921, and U.S. Pat. No. 6,380,248; seealso, e.g., U.S. Pat. No. 6,582,719) and several of these preparationsare able to inhibit biofilm formation.

BT compounds have proven activity against MRSA (methicillin resistant S.aureus), MRSE (methicillin resistant S. epidermidis), Mycobacteriumtuberculosis, Mycobacterium avium, drug-resistant P. aeruginosa,enterotoxigenic E. coli, enterohemorrhagic E. coli, Klebsiellapneumoniae, Clostridium difficile, Heliobacter pylori, Legionellapneumophila, Enterococcus faecalis, Enterobacter cloacae, Salmonellatyphimurium, Proteus vulgaris, Yersinia enterocolitica, Vibrio cholerae,and Shigella Flexneri (Domenico et al., 1997 Antimicrob. AgentsChemother. 41:1697-1703). There is also evidence of activity againstcytomegalovirus, herpes simplex virus type 1 (HSV-1) and HSV-2, andyeasts and fungi, such as Candida albicans. BT roles have also beendemonstrated in reducing bacterial pathogenicity, inhibiting or killinga broad spectrum of antibiotic-resistant microbes (gram-positive andgram-negative), preventing biofilm formation, preventing septic shock,treating sepsis, and increasing bacterial susceptibility to antibioticsto which they previously exhibited resistance (see, e.g., Domenico etal., 2001 Agents Chemother. 45:1417-1421; Domenico et al., 2000 Infect.Med. 17:123-127; Domenico et al., 2003 Res. Adv. In Antimicrob. Agents &Chemother. 3:79-85; Domenico et al., 1997 Antimicrob. Agents Chemother.41(8):1697-1703; Domenico et al., 1999 Infect. Immun. 67:664-669: Huanget al. 1999 J Antimicrob. Chemother. 44:601-605; Veloira et al., 2003 JAntimicrob. Chemother. 52:915-919; Wu et al., 2002 Am J Respir Cell Mol.Biol. 26:731-738).

Despite the availability of BT compounds for well over a decade,effective selection of appropriate BT compounds for particularinfectious disease indications has remained an elusive goal, wherebehavior of a particular BT against a particular microorganism cannot bepredicted, where synergistic activity of a particular BT and aparticular antibiotic against a particular microorganism cannot bepredicted, where BT effects in vitro may not always predict BT effectsin vivo, and where BT effects against planktonic (single-cell) microbialpopulations may not be predictive of BT effects against microbialcommunities, such as bacteria organized into a biofilm. Additionally,limitations in solubility, tissue permeability, bioavailability,biodistribution and the like may in the cases of some BT compoundshinder the ability to deliver clinical benefit safely and effectively.The presently disclosed invention embodiments address these needs andoffer other related advantages.

BRIEF SUMMARY

As disclosed herein, and without wishing to be bound by theory,according to certain embodiments described herein bismuth-thiol (BT)compounds may be used as antiseptic agents for use in the treatment of awide variety of clinical infectious diseases and conditions and inpersonal healthcare, while also decreasing the costs incurred for thetreatment of such infections, including savings that are realized byprevention or prophylaxis mediated at least in part by BTs.

Also, in certain embodiments there are contemplated formulations fortreating tissues and/or surfaces that contain bacterial biofilms orbacteria related to biofilm formation (e.g., bacteria that are capableof forming or otherwise promoting biofilms), which formulations compriseone or more BT compound and one or more antibiotic compound, asdescribed herein, where according to non-limiting theory, appropriatelyselected combinations of BT compound(s) and antibiotic(s) based on thepresent disclosure provide heretofore unpredicted synergy in theantibacterial (including anti-biofilm) effects of such formulations,and/or unpredicted enhancing effects, for prevention, prophylaxis and/ortherapeutically effective treatment against microbial infectionsincluding infections that contain bacterial biofilms.

Also provided herein are bismuth-thiol compositions comprisingsubstantially monodisperse microparticulate suspensions, and methods fortheir synthesis and use.

According to certain embodiments there is provided a method forprotecting a natural surface against one or more of a bacterialpathogen, a fungal pathogen and a viral pathogen, comprising contactingthe surface with an effective amount of a BT composition underconditions and for a time sufficient for one or more of: (i) preventionof infection of the surface by the bacterial, fungal or viral pathogen,(ii) inhibition of cell viability or cell growth of substantially allplanktonic cells of the bacterial, fungal or viral pathogen, (iii)inhibition of biofilm formation by the bacterial, fungal or viralpathogen, and (iv) inhibition of biofilm viability or biofilm growth ofsubstantially all biofilm-form cells of the bacterial, fungal or viralpathogen, wherein the BT composition comprises a plurality of solidmicroparticles that exhibit a unimodal size distribution when the BTcomposition is analyzed on a particle size analyzer and that comprise abismuth-thiol (BT) compound that has not been micronized, milled orsubjected to super-critical fluid processing, substantially all of saidmicroparticles having a volumetric mean diameter of from about 0.4 μm toabout 5 μm, wherein the BT compound comprises bismuth or a bismuth saltand a thiol-containing compound.

In certain further embodiments the bacterial pathogen comprises at leastone of: (i) one or more gram-negative bacteria; (ii) one or moregram-positive bacteria; (iii) one or more antibiotic-sensitive bacteria;(iv) one or more antibiotic-resistant bacteria; (v) a bacterial pathogenthat is selected from the group consisting of Staphylococcus aureus (S.aureus), MRSA (methicillin-resistant S. aureus), Staphylococcusepidermidis, MRSE (methicillin-resistant S. epidermidis), Mycobacteriumtuberculosis, Mycobacterium avium, Pseudomonas aeruginosa,drug-resistant P. aeruginosa, Escherichia coli, enterotoxigenic E. coli,enterohemorrhagic E. coli, Klebsiella pneumoniae, Clostridium difficile,Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis,methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae,Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica,Vibrio cholera, Shigella flexneri, vancomycin-resistant Enterococcus(VRE), Burkholderia cepacia complex, Francisella tularensis, Bacillusanthracis, Yersinia pestis, Pseudomonas aeruginosa, Streptococcuspneumonia, penicillin-resistant Streptococcus pneumonia, Escherichiacoli, Burkholderia cepacia, Bukholderia multivorans, Mycobacteriumsmegmatis and Acinetobacter baumannii.

In certain other further embodiments at least one of (a) the bacterialpathogen exhibits resistance to an antibiotic that is selected frommethicillin, vancomycin, naficilin, gentamicin, ampicillin,chloramphenicol, doxycycline and tobramycin, (b) the surface comprisesan epithelial tissue surface that is selected from epidermis, dermis,respiratory tract, gastrointestinal tract and glandular linings, (c) thestep of contacting is performed one or a plurality of times, (d) atleast one step of contacting comprises one of spraying, irrigating,dipping and painting the surface, (e) at least one step of contactingcomprises one of inhaling, ingesting and orally irrigating, (f) at leastone step of contacting comprises administering to a subject by a routethat is selected from topically, intraperitoneally, orally,parenterally, intravenously, intraarterially, transdermally,sublingually, subcutaneously, intramuscularly, transbuccally,intranasally, via inhalation, intraoccularly, intraauricularly,intraventricularly, subcutaneously, intraadiposally, intraarticularlyand intrathecally, and (g) the BT composition comprises one or more BTcompounds selected from BisBAL, BisEDT, Bis-dimercaprol, Bis-DTT,Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT,Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT,Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, andBis-EDT/2-hydroxy-1-propanethiol.

In another embodiment there is provided a method for protecting anatural surface against one or more of a bacterial pathogen, a fungalpathogen and a viral pathogen, comprising contacting the surface with aneffective amount of a BT composition under conditions and for a timesufficient for one or more of (i) prevention of infection of the surfaceby the bacterial, fungal or viral pathogen, (ii) inhibition of cellviability or cell growth of substantially all planktonic cells of thebacterial, fungal or viral pathogen, (iii) inhibition of biofilmformation by the bacterial, fungal or viral pathogen, and (iv)inhibition of biofilm viability or biofilm growth of substantially allbiofilm-form cells of the bacterial, fungal or viral pathogen, whereinthe BT composition comprises a plurality of solid microparticles thatexhibit a unimodal size distribution when the BT composition is analyzedon a particle size analyzer and that comprise a bismuth-thiol (BT)compound which comprises bismuth or a bismuth salt and athiol-containing compound, substantially all of said microparticleshaving a volumetric mean diameter of from about 0.4 μm to about 5 μm,wherein the BT compound, wherein at least one of (a) the bacterialpathogen exhibits resistance to an antibiotic that is selected frommethicillin, vancomycin, naficilin, gentamicin, ampicillin,chloramphenicol, doxycycline and tobramycin, (b) the surface comprisesan epithelial tissue surface that is selected from epidermis, dermis,respiratory tract, gastrointestinal tract and glandular linings, (c) thestep of contacting is performed one or a plurality of times, (d) atleast one step of contacting comprises one of spraying, irrigating,dipping and painting the surface, (e) at least one step of contactingcomprises one of inhaling, ingesting and orally irrigating, (f) at leastone step of contacting comprises administering to a subject by a routethat is selected from topically, intraperitoneally, orally,parenterally, intravenously, intraarterially, transdermally,sublingually, subcutaneously, intramuscularly, transbuccally,intranasally, via inhalation, intraoccularly, intraauricularly,intraventricularly, subcutaneously, intraadiposally, intraarticularlyand intrathecally, and (g) the BT composition comprises one or more BTcompounds selected from the group consisting of BisBAL, BisEDT,Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr,Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol, andwherein the method further comprises contacting the surface with atleast one of (i) a synergizing antibiotic and (ii) a cooperativeantimicrobial efficacy enhancing antibiotic, simultaneously orsequentially and in any order with respect to the step of contacting thesurface with the BT composition.

In certain further embodiments at least one of (a) the synergizingantibiotic or the cooperative antimicrobial efficacy enhancingantibiotic comprises an antibiotic that is selected from anaminoglycoside antibiotic, a carbapenem antibiotic, a cephalosporinantibiotic, a fluoroquinolone antibiotic, a glycopeptide antibiotic, alincosamide antibiotic, a penicillinase-resistant penicillin antibiotic,and an aminopenicillin antibiotic, and (b) the synergizing antibiotic orthe cooperative antimicrobial efficacy enhancing antibiotic is anaminoglycoside antibiotic that is selected from amikacin, arbekacin,gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin and apramycin. In certainother embodiments the method comprises overcoming antibiotic resistancewhere an antibiotic-resistant bacterial pathogen is present on thenatural surface. In certain further embodiments at least one of (a) thebacterial pathogen is selected from Staphylococcus aureus (S. aureus),MRSA (methicillin-resistant S. aureus), Staphylococcus epidermidis, MRSE(methicillin-resistant S. epidermidis), Mycobacterium tuberculosis,Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant P.aeruginosa, Escherichia coli, enterotoxigenic E. coli, enterohemorrhagicE. coli, Klebsiella pneumoniae, Clostridium difficile, Heliobacterpylori, Legionella pneumophila, Enterococcus faecalis,methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae,Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica,Vibrio cholera, Shigella flexneri, vancomycin-resistant Enterococcus(VRE), Burkholderia cepacia complex, Francisella tularensis, Bacillusanthracis, Yersinia pestis, Pseudomonas aeruginosa, Streptococcuspneumonia, penicillin-resistant Streptococcus pneumonia, Escherichiacoli, Burkholderia cepacia, Bukholderia multivorans, Mycobacteriumsmegmatis and Acinetobacter baumannii, (b) the bacterial pathogenexhibits resistance to an antibiotic that is selected from methicillin,vancomycin, naficilin, gentamicin, ampicillin, chloramphenicol,doxycycline, tobramycin, clindamicin and gatifloxacin, (c) the surfacecomprises an epithelial surface of a tissue that is selected from thegroup consisting of epidermis, dermis, respiratory tract,gastrointestinal tract and glandular linings, (d) the step of contactingis performed one or a plurality of times, (e) at least one step ofcontacting comprises one of spraying, irrigating, dipping, coating andpainting the surface, (f) at least one step of contacting comprises oneof inhaling, ingesting and orally irrigating, (g) at least one step ofcontacting comprises administering to a subject by a route that isselected from topically, intraperitoneally, orally, parenterally,intravenously, intraarterially, transdermally, sublingually,subcutaneously, intramuscularly, transbuccally, intranasally, viainhalation, intraoccularly, intraauricularly, intraventricularly,subcutaneously, intraadiposally, intraarticularly and intrathecally, (h)the BT composition comprises one or more BT compounds selected fromBisBAL, BisEDT, Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol,Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal,Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol, (i)the synergizing or enhancing antibiotic comprises an antibiotic that isselected from clindamicin, gatifloxacin, an aminoglycoside antibiotic, acarbapenem antibiotic, a cephalosporin antibiotic, a fluoroquinoloneantibiotic, a penicillinase-resistant penicillin antibiotic, and anaminopenicillin antibiotic, and (j) the synergizing or enhancingantibiotic is an aminoglycoside antibiotic that is selected fromamikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.

In another embodiment of the present invention there is provided anantiseptic composition for treating a natural surface that containsbacterial biofilm, comprising at least one of (1) a composition thatcomprises (a) at least one BT composition that comprises a plurality ofsolid microparticles that exhibit a unimodal size distribution when thecomposition is analyzed on a particle size analyzer and that comprise abismuth-thiol (BT) compound which comprises bismuth or a bismuth saltand a thiol-containing compound, substantially all of saidmicroparticles having a volumetric mean diameter of from about 0.4 μm toabout 5 μm; and (b) at least one antibiotic compound that is capable ofacting synergistically with, or enhancing, the BT compound, (2) acomposition that comprises (a) at least one BT composition thatcomprises a plurality of solid microparticles that exhibit a unimodalsize distribution when the composition is analyzed on a particle sizeanalyzer and that comprise a bismuth-thiol (BT) compound which comprisesbismuth or a bismuth salt and a thiol-containing compound, substantiallyall of said microparticles having a volumetric mean diameter of fromabout 0.4 μm to about 5 μm; and (b) at least one antibiotic compoundthat is capable of acting synergistically with, or enhancing, the BTcompound, wherein the antibiotic compound comprises an antibiotic thatis selected from methicillin, vancomycin, naficilin, gentamicin,ampicillin, chloramphenicol, doxycycline, tobramycin, clindamicin,gatifloxacin, cefazolin and an aminoglycoside antibiotic, and (3) thecomposition of (2) wherein the aminoglycoside antibiotic is selectedfrom amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.In certain further embodiments the BT compound is selected from BisBAL,BisEDT, Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE,Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol.

According to certain other embodiments there is provided a method fortreating a natural surface that contains bacterial biofilm, comprising(a) identifying a bacterial infection in or on the surface as comprisingone of (i) gram positive bacteria, (ii) gram negative bacteria, and(iii) both (i) and (ii); and (b) administering a formulation thatcomprises one or more bismuth thiol (BT) compositions to the surface,wherein (i) if the bacterial infection comprises gram positive bacteria,then the formulation comprises effective amounts of at least one BTcompound and at least one antibiotic that is rifamycin, (ii) if thebacterial infection comprises gram negative bacteria, then theformulation comprises effective amounts of at least one BT compound andamikacin, (iii) if the bacterial infection comprises both gram positiveand gram negative bacteria, then the formulation comprises effectiveamounts of one or a plurality of BT compounds, rifamycin and amikacin,and thereby treating the surface, wherein the BT compound comprisesbismuth or a bismuth salt and a thiol-containing compound. In certainfurther embodiments the bacterial infection comprises one or a pluralityof antibiotic-resistant bacteria. In certain further embodimentstreating comprises at least one of: (i) eradicating the bacterialbiofilm, (ii) reducing the bacterial biofilm, and (iii) impairing growthof the bacterial biofilm.

In certain other related embodiments the BT composition comprises aplurality of solid microparticles that exhibit a unimodal sizedistribution when the composition is analyzed on a particle sizeanalyzer and that comprise a bismuth-thiol (BT) compound, substantiallyall of said microparticles having a volumetric mean diameter of fromabout 0.4 μm to about 5 μm, wherein the BT compound comprises bismuth ora bismuth salt and a thiol-containing compound. In certain embodimentsthe BT compound has not been micronized, milled or subjected tosuper-critical fluid processing.

According to certain embodiments of the invention described herein thereis thus provided a bismuth-thiol composition, comprising a plurality ofmicroparticles that comprise a bismuth-thiol (BT) compound,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm, wherein the BT compoundcomprises bismuth or a bismuth salt and a thiol-containing compound. Inanother embodiment there is provided a bismuth-thiol composition,comprising a plurality of microparticles that comprise a bismuth-thiol(BT) compound, substantially all of said microparticles having avolumetric mean diameter of from about 0.4 μm to about 5 μm and beingformed by a process that comprises (a) admixing, under conditions andfor a time sufficient to obtain a solution that is substantially free ofa solid precipitate, (i) an acidic aqueous solution that comprises abismuth salt comprising bismuth at a concentration of at least 50 mM andthat lacks a hydrophilic, polar or organic solubilizer, with (ii)ethanol in an amount sufficient to obtain an admixture that comprises atleast about 5%, 10%, 15%, 20%, 25% or 30% ethanol by volume; and (b)adding to the admixture of (a) an ethanolic solution comprising athiol-containing compound to obtain a reaction solution, wherein thethiol-containing compound is present in the reaction solution at a molarratio of from about 1:3 to about 3:1 relative to the bismuth, underconditions and for a time sufficient for formation of a precipitatewhich comprises the microparticles comprising the BT compound. Incertain embodiments the bismuth salt is Bi(NO₃)₃. In certain embodimentsthe acidic aqueous solution comprises at least 5%, 10%, 15%, 20%, 22% or22.5% bismuth by weight. In certain embodiments the acidic aqueoussolution comprises at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%or 5% nitric acid by weight. In certain embodiments the thiol-containingcompound comprises one or more agents selected from 1,2-ethane dithiol,2,3-dimercaptopropanol, pyrithione, dithioerythritol,3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3-propanedithiol,2-hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol,alpha-lipoic acid and dithiothreitol.

In another embodiment there is provided a method for preparing abismuth-thiol composition that comprises a plurality of microparticlesthat comprise a bismuth-thiol (BT) compound, substantially all of saidmicroparticles having a volumetric mean diameter of from about 0.4 μm toabout 5 μm, said method comprising the steps of (a) admixing, underconditions and for a time sufficient to obtain a solution that issubstantially free of a solid precipitate, (i) an acidic aqueoussolution that comprises a bismuth salt comprising bismuth at aconcentration of at least 50 mM and that lacks a hydrophilic, polar ororganic solubilizer, with (ii) ethanol in an amount sufficient to obtainan admixture that comprises at least about 5%, 10%, 15%, 20%, 25% or 30%ethanol by volume; and (b) adding to the admixture of (a) an ethanolicsolution comprising a thiol-containing compound to obtain a reactionsolution, wherein the thiol-containing compound is present in thereaction solution at a molar ratio of from about 1:3 to about 3:1relative to the bismuth, under conditions and for a time sufficient forformation of a precipitate which comprises the microparticles comprisingthe BT compound. In certain embodiments the method further comprisesrecovering the precipitate to remove impurities. In certain embodimentsthe bismuth salt is Bi(NO₃)₃. In certain embodiments the acidic aqueoussolution comprises at least 5%, 10%, 15%, 20%, 22% or 22.5% bismuth byweight. In certain embodiments the acidic aqueous solution comprises atleast 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% nitric acid byweight. In certain embodiments the thiol-containing compound comprisesone or more agents selected from the group consisting of 1,2-ethanedithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol,3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3-propanedithiol,2-hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol,dithiothreitol, alpha-lipoic acid, methanethiol (CH₃SH [m-mercaptan]),ethanethiol (C₂H₅SH [e-mercaptan]), 1-propanethiol (C₃H₇SH [n-Pmercaptan]), 2-propanethiol (CH₃CH(SH)CH₃ [2C₃ mercaptan]), butanethiol(C₄H₉SH ([n-butyl mercaptan]), tert-butyl mercaptan (C(CH₃)₃SH [t-butylmercaptan]), pentanethiols (C₅H₁₁SH [pentyl mercaptan]), coenzyme A,lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol,dithiothreitol, dithioerythritol, 2-mercaptoindole, transglutaminase,(11-mercaptoundecyl)hexa(ethylene glycol),(11-mercaptoundecyl)tetra(ethylene glycol),(11-mercaptoundecyl)tetra(ethylene glycol) functionalized goldnanoparticles, 1,1′,4′,1″-terphenyl-4-thiol, 1,11-undecanedithiol,1,16-hexadecanedithiol, 1,2-ethanedithiol technical grade,1,3-propanedithiol, 1,4-benzenedimethanethiol, 1,4-butanedithiol,1,4-butanedithiol diacetate, 1,5-pentanedithiol, 1,6-hexanedithiol,1,8-octanedithiol, 1,9-nonanedithiol, adamantanethiol, 1-butanethiol,1-decanethiol, 1-dodecanethiol, 1-heptanethiol, 1-heptanethiol purum,1-hexadecanethiol, 1-hexanethiol, 1-mercapto-(triethylene glycol),1-mercapto-(triethylene glycol) methyl ether functionalized goldnanoparticles, 1-mercapto-2-propanol, 1-nonanethiol, 1-octadecanethiol,1-octanethiol, 1-octanethiol, 1-pentadecanethiol, 1-pentanethiol,1-propanethiol, 1-tetradecanethiol, 1-tetradecanethiol purum,1-undecanethiol, 11-(1H-pyrrol-1-yl)undecane-1-thiol,11-amino-1-undecanethiol hydrochloride, 11-bromo-1-undecanethiol,11-mercapto-1-undecanol, 11-mercapto-1-undecanol, 11-mercaptoundecanoicacid, 11-mercaptoundecanoic acid, 11-mercaptoundecyl trifluoroacetate,11-mercaptoundecylphosphoric acid, 12-mercaptododecanoic acid,12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid,16-mercaptohexadecanoic acid, 16-mercaptohexadecanoic acid,1H,1H,2H,2H-perfluorodecanethiol, 2,2′-(ethylenedioxy)diethanethiol,2,3-butanedithiol, 2-butanethiol, 2-ethylhexanethiol,2-methyl-1-propanethiol, 2-methyl-2-propanethiol, 2-phenylethanethiol,3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanethiolpurum,3-(dimethoxymethylsilyl)-1-propanethiol, 3-chloro-1-propanethiol,3-mercapto-1-propanol, 3-mercapto-2-butanol,3-mercapto-N-nonylpropionamide, 3-mercaptopropionic acid,3-mercaptopropyl-functionalized silica gel, 3-methyl-1-butanethiol,4,4′-bis(mercaptomethyl)biphenyl, 4,4′-dimercaptostilbene,4-(6-mercaptohexyloxyl)benzyl alcohol, 4-cyano-1-butanethiol,4-mercapto-1-butanol, 6-(ferrocenyl)hexanethiol, 6-mercapto-1-hexanol,6-mercaptohexanoic acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid,9-mercapto-1-nonanol, biphenyl-4,4′-dithiol, butyl 3-mercaptopropionate,copper(I) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol,decanethiol functionalized silver nanoparticles, dodecanethiolfunctionalized gold nanoparticles, dodecanethiol functionalized silvernanoparticles, hexa(ethylene glycol)mono-11-(acetylthio)undecyl ether,mercaptosuccinic acid, methyl 3-mercaptopropionate, heptanoic hydrazidebisphenol A substituted5-(Hydroxymethyl)-1:3-bis(6-mercaptohexyloxy)benzene sold under thetrademark NANO TETHER BPA-HH™ (Sigma-Aldrich, St. Louis, Mo.),octadecanethiol reagents sold under the trademark NANOTHINKS™ 18(Sigma-Aldrich) octanethiol reagents sold under the trademarkNANOTHINKS™ 8 (Sigma-Aldrich), 11-mercaptoundecanoic acid reagents soldunder the trademark NANOTHINKS™ ACID11 (Sigma-Aldrich),16-mercaptorhexadecanoic acid reagents sold under the trademarkNANOTHINKS™ ACID16 (Sigma-Aldrich), 11-mercapto-1-undecanol reagentssold under the trademark NANOTHINKS™ ALCO11 (Sigma-Aldrich),1,8-octanedithiol sold under the trademark NANOTHINKS™ THIO8(Sigma-Aldrich), octanethiol functionalized gold nanoparticles, PEGdithiol average M_(n) 8,000, PEG dithiol average mol wt 1,500, PEGdithiol average mol wt 3,400, S-(11-bromoundecyl)thioacetate,S-(4-cyanobutyl)thioacetate, thiophenol, triethylene glycolmono-11-mercaptoundecyl ether, trimethylolpropanetris(3-mercaptopropionate),[11-(methylcarbonylthio)undecyl]tetra(ethylene glycol),m-carborane-9-thiol, p-terphenyl-4,4″-dithiol, tert-dodecylmercaptan,tert-nonyl mercaptan.

In another embodiment there is provided a method for protecting anatural surface, including a biological tissue surface such as anepithelial tissue surface, against one or more of a bacterial pathogen,a fungal pathogen and a viral pathogen, comprising contacting theepithelial tissue surface with an effective amount of a BT compositionunder conditions and for a time sufficient for one or more of (i)prevention of infection of the surface by the bacterial, fungal or viralpathogen, (ii) inhibition of cell viability or cell growth ofsubstantially all planktonic cells of the bacterial, fungal or viralpathogen, (iii) inhibition of biofilm formation by the bacterial, fungalor viral pathogen, and (iv) inhibition of biofilm viability or biofilmgrowth of substantially all biofilm-form cells of the bacterial, fungalor viral pathogen, wherein the BT composition comprises a plurality ofmicroparticles that comprise a bismuth-thiol (BT) compound,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm. In certain embodiments thebacterial pathogen comprises at least one of (i) one or moregram-negative bacteria; (ii) one or more gram-positive bacteria; (iii)one or more antibiotic-sensitive bacteria; (iv) one or moreantibiotic-resistant bacteria; (v) a bacterial pathogen that is selectedfrom Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant S.aureus), Staphylococcus epidermidis, MRSE (methicillin-resistant S.epidermidis), Mycobacterium tuberculosis, Mycobacterium avium,Pseudomonas aeruginosa, drug-resistant P. aeruginosa, Escherichia coli,enterotoxigenic E. coli, enterohemorrhagic E. coli, Klebsiellapneumoniae, Clostridium difficile, Heliobacter pylori, Legionellapneumophila, Enterococcus faecalis, methicillin-susceptible Enterococcusfaecalis, Enterobacter cloacae, Salmonella typhimurium, Proteusvulgaris, Yersinia enterocolitica, Vibrio cholera, Shigella flexneri,vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia complex,Francisella tularensis, Bacillus anthracis, Yersinia pestis, Pseudomonasaeruginosa, vancomycin-resistant enterococci, Streptococcus pneumonia,penicillin-resistant Streptococcus pneumonia, Escherichia coli,Burkholderia cepacia, Bukholderia multivorans, Mycobacterium smegmatisand Acinetobacter baumannii. In certain embodiments the bacterialpathogen exhibits antibiotic resistance. In certain embodiments thebacterial pathogen exhibits resistance to an antibiotic that is selectedfrom methicillin, vancomycin, naficilin, gentamicin, ampicillin,chloramphenicol, doxycycline and tobramycin.

In certain embodiments the natural surface comprises an oral/buccalcavity surface. In further embodiments, the natural surface comprises abiological surface such as bone, joint, muscle, ligament, or tendon.

In certain embodiments the surface comprises an epithelial tissuesurface that comprises a tissue that is selected from epidermis, dermis,respiratory tract, gastrointestinal tract and glandular linings.

In certain embodiments the step of contacting is performed one or aplurality of times. In certain embodiments at least one step ofcontacting comprises one of spraying, irrigating, dipping and paintingthe natural surface. In certain embodiments at least one step ofcontacting comprises one of inhaling, ingesting and orally irrigating.In certain embodiments least one step of contacting comprisesadministering by a route that is selected from topically,intraperitoneally, orally, parenterally, intravenously, intraarterially,transdermally, sublingually, subcutaneously, intramuscularly,transbuccally, intranasally, via inhalation, intraoccularly,intraauricularly, intraventricularly, subcutaneously, intraadiposally,intraarticularly and intrathecally. In certain embodiments the BTcomposition comprises one or more BT compounds selected from the groupconsisting of BisBAL, BisEDT, Bis-dimercaprol, Bis-DTT,Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT,Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT,Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, andBis-EDT/2-hydroxy-1-propanethiol.

In certain embodiments the bacterial pathogen exhibits antibioticresistance. In certain other embodiments the above described methodfurther comprises contacting the natural surface with a synergizingantibiotic and/or with an enhancing antibiotic, simultaneously orsequentially and in any order with respect to the step of contacting thesurface with the BT composition. In certain embodiments the synergizingand/or enhancing antibiotic comprises an antibiotic that is selectedfrom an aminoglycoside antibiotic, a carbapenem antibiotic, acephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptideantibiotic, a lincosamide antibiotic, a penicillinase-resistantpenicillin antibiotic, and an aminopenicillin antibiotic. In certainembodiments the synergizing and/or enhancing antibiotic is anaminoglycoside antibiotic that is selected from amikacin, arbekacin,gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin and apramycin.

In another embodiment of the invention described herein there isprovided a method for overcoming antibiotic resistance (e.g., for abacterial pathogen that is resistant to at least one anti-bacterialeffect of at least one antibiotic known to have an anti-bacterial effectagainst bacteria of the same bacterial species, rendering such apathogen susceptible to an antibiotic) on a natural surface where anantibiotic-resistant bacterial pathogen is present, comprisingcontacting the surface simultaneously or sequentially and in any orderwith an effective amount of (1) at least one bismuth-thiol (BT)composition and (2) at least one antibiotic that is enhanced by, and/orthat is capable of acting synergistically with the at least one BTcomposition, under conditions and for a time sufficient for one or moreof: (i) prevention of infection of the surface by the bacterialpathogen, (ii) inhibition of cell viability or cell growth ofsubstantially all planktonic cells of the bacterial pathogen, (iii)inhibition of biofilm formation by the bacterial pathogen, and (iv)inhibition of biofilm viability or biofilm growth of substantially allbiofilm-form cells of the bacterial pathogen, wherein the BT compositioncomprises a plurality of microparticles that comprise a bismuth-thiol(BT) compound, substantially all of said microparticles having avolumetric mean diameter of from about 0.4 μm to about 5 μm; and therebyovercoming antibiotic resistance on the epithelial tissue surface. Incertain embodiments the bacterial pathogen comprises at least one of:(i) one or more gram-negative bacteria; (ii) one or more gram-positivebacteria; (iii) one or more antibiotic-sensitive bacteria; (iv) one ormore antibiotic-resistant bacteria; (v) a bacterial pathogen that isselected from Staphylococcus aureus (S. aureus), MRSA(methicillin-resistant S. aureus), Staphylococcus epidermidis, MRSE(methicillin-resistant S. epidermidis), Mycobacterium tuberculosis,Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant P.aeruginosa, Escherichia coli, enterotoxigenic E. coli, enterohemorrhagicE. coli, Klebsiella pneumoniae, Clostridium difficile, Heliobacterpylori, Legionella pneumophila, Enterococcus faecalis,methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae,Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica,Vibrio cholera, Shigella flexneri, vancomycin-resistant Enterococcus(VRE), Burkholderia cepacia complex, Francisella tularensis, Bacillusanthracis, Yersinia pestis, Pseudomonas aeruginosa, vancomycin-resistantenterococci, Streptococcus pneumonia, penicillin-resistant Streptococcuspneumonia, Escherichia coli, Burkholderia cepacia, Bukholderiamultivorans, Mycobacterium smegmatis and Acinetobacter baumannii.

In certain embodiments the bacterial pathogen exhibits resistance to anantibiotic that is selected from methicillin, vancomycin, naficilin,gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin,clindamicin and gatifloxacin.

In certain embodiments the natural surface comprises an oral/buccalcavity surface. In further embodiments, the natural surface comprises abiological surface such as bone, joint, muscle, ligament, or tendon.

In certain embodiments the surface comprises a tissue that is selectedfrom the group consisting of epidermis, dermis, respiratory tract,gastrointestinal tract and glandular linings. In certain embodiments thestep of contacting is performed one or a plurality of times. In certainembodiments at least one step of contacting comprises one of spraying,irrigating, dipping and painting the surface. In certain otherembodiments at least one step of contacting comprises one of inhaling,ingesting and orally irrigating. In certain embodiments at least onestep of contacting comprises administering by a route that is selectedfrom topically, intraperitoneally, orally, parenterally, intravenously,intraarterially, transdermally, sublingually, subcutaneously,intramuscularly, transbuccally, intranasally, via inhalation,intraoccularly, intraauricularly, intraventricularly, subcutaneously,intraadiposally, intraarticularly and intrathecally. In certainembodiments the BT composition comprises one or more BT compoundsselected from BisBAL, BisEDT, Bis-dimercaprol, Bis-DTT,Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT,Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT,Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, andBis-EDT/2-hydroxy-1-propanethiol. In certain embodiments the synergizingand/or enhancing antibiotic comprises an antibiotic that is selectedfrom clindamicin, gatifloxacin, an aminoglycoside antibiotic, acarbapenem antibiotic, a cephalosporin antibiotic, a fluoroquinoloneantibiotic, a glycopeptide antibiotic, a lincosamide antibiotic, apenicillinase-resistant penicillin antibiotic, and an aminopenicillinantibiotic. In certain embodiments the synergizing and/or enhancingantibiotic is an aminoglycoside antibiotic that is selected fromamikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.

Turning to another embodiment there is provided an antisepticcomposition, comprising (a) at least one BT compound; (b) at least oneantibiotic compound that is enhanced by and/or is capable of actingsynergistically with the BT compound; and (c) a pharmaceuticallyacceptable excipient or carrier, including a carrier for topical use. Incertain embodiments the BT compound is selected from BisBAL, BisEDT,Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr,Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol. Incertain embodiments the BT composition comprises a plurality ofmicroparticles that comprise a bismuth-thiol (BT) compound,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm. In certain embodiments theBT compound is selected from BisEDT and BisBAL. In certain embodimentsthe antibiotic compound comprises an antibiotic that is selected frommethicillin, vancomycin, naficilin, gentamicin, ampicillin,chloramphenicol, doxycycline, tobramycin, clindamicin, gatifloxacin andan aminoglycoside antibiotic. In certain embodiments the aminoglycosideantibiotic is selected from amikacin, arbekacin, gentamicin, kanamycin,neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin,tobramycin and apramycin. In certain embodiments the aminoglycosideantibiotic is amikacin.

In certain other embodiments there is provided a method for treating anatural surface that supports or contains bacterial biofilm, comprising(a) identifying a bacterial infection on or in the surface as comprisingone of (i) gram positive bacteria, (ii) gram negative bacteria, and(iii) both (i) and (ii); and (b) administering a formulation thatcomprises one or more bismuth thiol (BT) compositions to the surface,wherein (i) if the bacterial infection comprises gram positive bacteria,then the formulation comprises therapeutically effective amounts of atleast one BT compound and at least one antibiotic that is rifamycin,(ii) if the bacterial infection comprises gram negative bacteria, thenthe formulation comprises therapeutically effective amounts of at leastone BT compound and amikacin, (iii) if the bacterial infection comprisesboth gram positive and gram negative bacteria, then the formulationcomprises therapeutically effective amounts of one or a plurality of BTcompounds, rifamycin and amikacin, and thereby treating the surface.

In certain embodiments the biofilm comprises one or a plurality ofantibiotic-resistant bacteria. In certain embodiments treating thesurface comprises at least one of: (i) eradicating the bacterialbiofilm, (ii) reducing the bacterial biofilm, and (iii) impairing growthof the bacterial biofilm. In certain embodiments the BT compositioncomprises a plurality of microparticles that comprise a bismuth-thiol(BT) compound, substantially all of said microparticles having avolumetric mean diameter of from about 0.4 μm to about 5 μm.

These and other aspects of the herein described invention embodimentswill be evident upon reference to the following detailed description andattached drawings. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, includingU.S. RE37,793, U.S. Pat. No. 6,248,371, U.S. Pat. No. 6,086,921, andU.S. Pat. No. 6,380,248, are incorporated herein by reference in theirentirety, as if each was incorporated individually. Aspects andembodiments of the invention can be modified, if necessary, to employconcepts of the various patents, applications and publications toprovide yet further embodiments.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows surviving numbers (log CFU; colony forming units) fromPseudomonas aeruginosa colony biofilms grown for 24 hours on 10% trypticsoy agar (TSA) at 37° C., followed with indicated treatment for 18hours. Indicated antibiotic treatments are TOB, tobramycin 10×MIC; AMK,amikacin 100×MIC; IPM, imipenem 10×MIC; CEF, cefepime 10×MIC; CIP,ciprofloxacin 100×MIC; Cpd 2B, compound 2B (Bis-BAL, 1:1.5). (MIC;minimum inhibitory concentration, e.g., lowest concentration thatprevents bacterial growth).

FIG. 2 shows surviving numbers (log CFU) from Staphylococcus aureuscolony biofilms grown for 24 hours on 10% tryptic soy agar, followed bythe indicated treatment. Indicated antibiotic treatments are Rifampicin,RIF 100×MIC; daptomycin, DAP 320×MIC; minocycline, MIN 100×MIC;ampicillin, AMC 10×MIC; vancomycin, VAN 10×MIC; Cpd 2B, compound 2B(Bis-BAL, 1:1.5), Cpd 8-2, compound 8-2 (Bis-Pyr/BDT (1:1/0.5).

FIG. 3 shows scratch closure over time of keratinocytes exposed tobiofilms. (*) Significantly different from control (P<0.001).

FIGS. 4A and 4B show the subinhibitory BisEDT reversingantibiotic-resistance to several antibiotics. Effects of antibioticswith and without BisEDT (0.05 μg/ml) on a lawn of MRSA(Methicillin-resistant S. aureus) is shown. Panel A shows standardantibiotic-soaked discs alone, and Panel B shows discs combined with aBisEDT (BE). [GM=gentamicin, CZ=cefazolin, FEP=cefepime,IPM=imipenim,SAM=ampicillin/sulbactam, LVX=levofloxacin.

FIG. 5 shows the effect of BisEDT and antibiotics on biofilm formation.S. epidermidis grown in TSB+2% glucose in polystyrene plates for 48 h at37° C. Gatifloxacin (GF), clindamycin (CM), minocycline (MC), gentamicin(GM), vancomycin (VM), cefazolin (CZ), nafcillin (NC), and rifampicin(RP). Results were expressed as the mean change in the BPC (in serial2-fold dilution steps) at 0.25 μM BisEDT (n=3).

FIG. 6 shows the effect of BisEDT and antibiotics on growth of S.epidermidis grown in TSB plus 2% glucose for 48 h at 37° C. Results areexpressed as the mean change in MIC (dilution steps) with increasingBisEDT (n=3). See legend in FIG. 5 for antibiotic definitions.

FIG. 7 is a bar graph showing the mean S. aureus bacteria levelsdetected on the bone and hardware samples from open fractures in an invivo rat model following treatment with three BT formulations, Bis-EDT,MB-11 and MB-8-2 with or without Cefazolin antibiotic treatment.Standard errors of the mean are shown as error bars. Animals euthanizedearly are not excluded from the analysis, however samples from oneanimal in group 2 have been excluded due to gross contamination.

DETAILED DESCRIPTION

Particular embodiments of the invention disclosed herein are based onthe surprising discovery that certain bismuth-thiol (BT) compounds asprovided herein (in certain embodiments including BT microparticleshaving a volumetric mean diameter of from about 0.4 μm to about 5 μm),but not certain other BT compounds (even if provided as microparticles),exhibited potent antiseptic, antibacterial and/or anti-biofilm activityagainst particular bacteria, including bacteria associated with a numberof clinically significant infections including infections that cancomprise bacterial biofilms.

Unexpectedly, not all BT compounds were uniformly effective against suchbacteria in a predictable fashion, but instead exhibited differentpotencies depending on the target bacterial species. In particular andas described herein, certain BT compounds (preferably including BTmicroparticles having a volumetric mean diameter of from about 0.4 μm toabout 5 μm) were found to exhibit higher potency against gram-negativebacteria, while certain other BT compounds (preferably including BTmicroparticles having a volumetric mean diameter of from about 0.4 μm toabout 5 μm) were found to exhibit greater potency against gram-positivebacteria, in a manner that, according to non-limiting theory, may forthe first time afford clinically relevant strategies for the managementof bacterial infections, including bacterial biofilm infections.

Additionally, and as described in greater detail below, certainembodiments of the invention described herein relate to surprisingadvantages that are provided by novel bismuth-thiol (BT) compositionsthat, as disclosed herein, can be made in preparations that comprise aplurality of BT microparticles that are substantially monodisperse withrespect to particle size (e.g., having volumetric mean diameter fromabout 0.4 μm to about 5 μm). In certain of these and relatedembodiments, the microparticulate BT is not provided as a component of alipid vesicle or liposome such as a multilamellarphosphocholine-cholesterol liposome or other multilamellar orunilamellar liposomal vesicle.

As also disclosed herein, with respect to certain embodiments, it hasbeen discovered that antibacterial and anti-biofilm efficacies ofcertain antibiotics, which antibiotics have previously been found tolack potent therapeutic effect against such bacterial infections, may besignificantly enhanced (e.g., increased in a statistically significantmanner) by treating the infection (e.g., by direct application on or inan infected site such as a natural surface) with one or more of theseantibiotics in concert, simultaneously or sequentially and in eitherorder, with a selected BT compound. In a manner that could not bepredicted prior to the present disclosure, certain BT compounds can becombined with certain antibiotics to provide a synergizing or enhancingcombination as provided herein with respect to antibacterial and/oranti-biofilm activity against certain bacterial species or bacterialstrains. The unpredicted nature of such combinations, as described ingreater detail below, is evidenced by the observations that whilecertain BT/antibiotic combinations acted synergistically or exhibitedenhancement against certain bacteria, certain other BT/antibioticcombinations failed to exhibit such synergistic or enhancedantibacterial and/or anti-biofilm activity.

According to these and related embodiments, the antibiotic and the BTcompound may be administered simultaneously or sequentially and ineither order, and it is noteworthy that the specific synergizing orenhancing combinations of one or more antibiotic and one or more BTcompound as disclosed herein for treatment of a particular infection(e.g., a biofilm formed by gram-negative or gram-positive bacteria) didnot exhibit predictable (e.g., merely additive) activities but insteadacted in an unexpectedly synergistic or enhancing (e.g., supra-additive)fashion, as a function of the selected antibiotic, the selected BTcompound and the specifically identified target bacteria.

For example, by way of illustration and not limitation, disclosed hereinin the context of a wide variety of actually or potentially microbiallyinfected natural surfaces, and further in the context of improvedsubstantially monodisperse microparticulate BT formulations, either orboth of a particular antibiotic compound and a particular BT compoundmay exert limited antibacterial effects when used alone against aparticular bacterial strain or species, but the combination of both theantibiotic compound and the BT compound exerts a potent antibacterialeffect against the same bacterial strain or species, which effect isgreater in magnitude (with statistical significance) than the simple sumof the effects of each compound when used alone, and is thereforebelieved according to non-limiting theory to reflect antibiotic-BTsynergy (e.g., FICI≦0.5) or an enhancing effect (e.g., 0.5<FICI≦1.0) ofthe BT on the antibiotic potency and/or of the antibiotic on the BTpotency. Accordingly, not every BT compound may synergize with, or beenhancing for, every antibiotic, and not every antibiotic may synergizewith, or be enhancing for, every BT compound, such that antibiotic-BTsynergy and BT-antibiotic enhancement generally are not predictable.Instead, and according to certain embodiments as disclosed herein,specific combinations of synergizing or enhancing antibiotic and BTcompounds surprisingly confer potent antibacterial effects againstparticular bacteria, including in particular environments such asnatural surfaces as described herein, and further including in certainsituations antibacterial effects against biofilms formed by theparticular bacteria.

That is, certain BT-synergizing antibiotics are described herein, whichincludes an antibiotic that is capable of acting synergistically(FICI≦0.5) with at least one BT composition that comprises at least oneBT compound as provided herein, where such synergy manifests as adetectable effect that is greater (i.e., in a statistically significantmanner relative to an appropriate control condition) in magnitude thanthe effect that can be detected when the antibiotic is present but theBT compound is absent, and/or when the BT compound is present but theantibiotic is absent. Similarly, certain BT-antibiotic combinationsexhibit enhancement (0.5<FICI≦1.0), where such enhancement manifests asa detectable effect that is greater (i.e., in a statisticallysignificant manner relative to an appropriate control condition) inmagnitude than the effect that can be detected when the antibiotic ispresent but the BT compound is absent, and/or when the BT compound ispresent but the antibiotic is absent.

Examples of such a detectable effect may in certain embodiments include(i) prevention of infection by a bacterial pathogen, (ii) inhibition ofcell viability or cell growth of substantially all planktonic cells of abacterial pathogen, (iii) inhibition of biofilm formation by a bacterialpathogen, and (iv) inhibition of biofilm viability or biofilm growth ofsubstantially all biofilm-form cells of a bacterial pathogen, but theinvention is not intended to be so limited, such that in othercontemplated embodiments antibiotic-BT synergy may manifest as one ormore detectable effects that may include alteration (e.g., astatistically significant increase or decrease) of one or more otherclinically significant parameters, for example, the degree of resistanceor sensitivity of a bacterial pathogen to one or more antibiotics orother drugs or chemical agents, the degree of resistance or sensitivityof a bacterial pathogen to one or more chemical, physical or mechanicalconditions (e.g., pH, ionic strength, temperature, pressure), and/or thedegree of resistance or sensitivity of a bacterial pathogen to one ormore biological agents (e.g., a virus, another bacterium, a biologicallyactive polynucleotide, an immunocyte or an immunocyte product such as anantibody, cytokine, chemokine, enzyme including degradative enzymes,membrane-disrupting protein, a free radical such as a reactive oxygenspecies, or the like).

Persons familiar with the art will appreciate these and a variety ofother criteria by which the effects of particular agents on thestructure, function and/or activity of a bacterial population may bedetermined (e.g., Coico et al. (Eds.), Current Protocols inMicrobiology, 2008, John Wiley & Sons, Hoboken, N.J.; Schwalbe et al.,Antimicrobial Susceptibility Testing Protocols, 2007, CRC Press, BocaRaton, Fla.), for purposes of ascertaining antibiotic-BT synergy orenhancement which, as provided herein, is present when the effects ofthe synergizing or enhancing antibiotic-BT combination exceed the meresum of the effects observed when one component of the combination is notpresent.

For example, in certain embodiments synergy may be determined bydetermining an antibacterial effect such as those described herein usingvarious concentrations of candidate agents (e.g., a BT and an antibioticindividually and in combination) to calculate a fractional inhibitoryconcentration index (FICI) and a fractional bactericidal concentrationindex (FBCI), according to Eliopoulos et al. (Eliopoulos and Moellering,(1996) Antimicrobial combinations. In Antibiotics in Laboratory Medicine(Lorian, V., Ed.), pp. 330-96, Williams and Wilkins, Baltimore, Md.,USA). Synergy may be defined as an FICI or FBCI index of ≦0.5, andantagonism at >4. (e.g., Odds, F C (2003) Synergy, antagonism, and whatthe chequerboard puts between them. Journal of AntimicrobialChemotherapy 52:1). Synergy may also be defined conventionally as≧4-fold decrease in antibiotic concentration, or alternatively, usingfractional inhibitory concentration (FIC) as described, e.g., byHollander et al. (1998 Antimicrob. Agents Chemother. 42:744). In certainembodiments, synergy may be defined as an effect that results from acombination of two drugs (e.g., an antibiotic and a BT composition)wherein the effect of the combination is greater (e.g., in astatistically significant manner) than it would be if the concentrationof the second drug is replaced by the first drug.

Accordingly as described herein and in certain preferred embodiments, acombination of BT and antibiotic will be understood to synergize when aFICI value that is less than or equal to 0.5 is observed. (Odds, 2003).As also described herein, in certain other preferred embodiments andaccording to non-limiting theory, it is disclosed that certainBT-antibiotic combinations may exhibit a FICI value between 0.5 and 1.0that signifies a high potential for such synergy, and which may beobserved using non-optimal concentrations of at least one BT and atleast one antibiotic that exhibit unilateral or mutually enhancedcooperative antimicrobial efficacy. Such an effect may also be referredto herein as “enhanced” antibiotic activity or “enhanced” BT activity.

Enhanced antibiotic and/or BT activity may be detected according tocertain embodiments when the presence both (i) of at least one BT at aconcentration that is less (in a statistically significant manner) thanthe characteristic minimum inhibitory concentration (MIC) for that BTfor a given target microbe (e.g., a given bacterial species or strain),and (ii) of at least one antibiotic at a concentration that is less (ina statistically significant manner) than the characteristic IC₅₀(concentration that inhibits the growth of 50% of a microbialpopulation; e.g., Soothill et al., 1992 J Antimicrob Chemother29(2):137) and/or that is less than the biofilm-prevention concentration(BPC) of that antibiotic for the given target microbe, results inenhanced (in a statistically significant manner) antimicrobial efficacyof the BT-antibiotic combination relative to the antimicrobial effectthat would be observed if either antimicrobial agent (e.g., the BT orthe antibiotic) were used at the same concentration in the absence ofthe other antimicrobial agent (e.g., the antibiotic or the BT). Inpreferred embodiments, “enhanced” antibiotic and/or BT activity ispresent when a FICI value that is less than or equal to 1.0, and greaterthan 0.5, is determined.

As will be appreciated by the skilled person based on the presentdisclosure, in certain embodiments synergistic or enhanced antibioticand/or BT activity may be determined according to methods known in theart, such as using Loewe additivity-based models (e.g., FIC index, Grecomodel), or Bliss independence based models (e.g., non-parametric andsemi-parametric models) or other methods described herein and known inthe art (e.g., Meletiadis et al., 2005 Medical Mycology 43:133-152).Illustrative methods for determining synergy or enhanced antibioticand/or BT activity are thus described, for instance, in Meletiadis etal., 2005 Medical Mycology 43:133-152 and references cited therein (seealso, Meletiadis et al., 2002 Rev Med Microbiol 13:101-117; White etal., 1996 Antimicrob Agents Chemother 40:1914-1918; Mouton et al., 1999Antimicrob Agents Chemother 43:2473-2478).

Certain other embodiments contemplate specific combinations of one ormore antibiotic and one or more BT compound as disclosed herein that mayexhibit synergizing or enhancing effects in vivo for treatment of aparticular infection (e.g., a biofilm formed by gram-negative orgram-positive bacteria), even where the BT compound(s) and antibiotic(s)did not exhibit predictable (e.g., merely additive) activities in vivobut instead acted in an unexpectedly synergistic or enhancing (e.g.,supra-additive; or conferring an effect when two or more such agents arepresent in combination that is greater (e.g., in a statisticallysignificant manner) than the effect that is obtained if theconcentration of the second agent is replaced by the first agent)fashion, as a function of the selected antibiotic, the selected BTcompound and one or more of the specifically identified target bacterialspecies of which the infection is comprised. It will therefore beappreciated, according to these and related embodiments, that in certainin vivo situations FICI or FBCI values (which are determined in vitro)may not be readily available, but that instead BT-antibiotic synergizingor enhancing effects may be determined in a manner afforded by thequantifiable metrics of the infection.

For example, in one embodiment, such as in the in vivo open fractureRattus norvegicus femur critical defect model as described in Example11, a statistically significant reduction in bacterial counts observedpost-treatment for the BT-antibiotic combination as compared to theantibiotic treatment or BT compound alone, is an indication ofsynergizing or enhancing effects. Statistical significance can bedetermined using methods well-known to the skilled person. In certainother embodiments, a reduction observed in this or other in vivo modelsby at least 5%, 10%, 20%, 30%, 40%, or 50% of bacterial counts observedin the injury post-treatment for the BT-antibiotic combination ascompared to the antibiotic treatment or BT compound alone is consideredan indication of synergizing or enhancing effects.

Other exemplary indicia of in vivo infections may be determinedaccording to established methodologies that have been developed forquantification of the severity of the infection, such as a variety ofwound scoring systems known to the skilled person (see e.g., scoringsystems reviewed in European Wound Management Association (EWMA),Position Document: Identifying criteria for wound infection. London: MEPLtd, 2005). Illustrative wound scoring systems that may be used inassessing synergistic or enhancement activity of BT-antibioticcombinations as described herein include ASEPSIS (Wilson A P, J HospInfect 1995; 29(2): 81-86; Wilson et al., Lancet 1986; 1: 311-13), theSouthampton Wound Assessment Scale (Bailey I S, Karran S E, Toyn K, etal. BMJ 1992; 304: 469-71). See also, Horan T C, Gaynes P, Martone W J,et al., 1992 Infect Control Hosp Epidemiol 1992; 13: 606-08.Additionally, recognized clinical indicia of wound healing known to theskilled clinician may also be measured in the presence or absence of BTcompounds and/or antibiotics, such as wound size, depth, granulationtissue condition, infection, etc. Accordingly, and based on the presentdisclosure, the skilled person will readily appreciate a variety ofmethods for determining whether a BT composition—antibiotic combinationalters (e.g., increases or decreases in a statistically significantmanner relative to appropriate controls) in vivo wound healing.

In view of these and related embodiments, there are provided herein awide variety of methods for treating microbially infected naturalsurfaces such as surfaces that support or contain bacterial biofilms,with an effective amount (e.g., in certain embodiments a therapeuticallyeffective amount) of a composition or formulation that comprises one ormore BT compounds and, optionally, one or more antibiotic compounds,such as one or more synergizing antibiotics, or one or more enhancingantibiotics, as provided herein. It will be appreciated that based onthe present disclosure, certain antibiotics are now contemplated for usein the treatment of given types of infections, where such antibioticshad previously been viewed by persons familiar with the art asineffective against infections of the same type.

Certain embodiments thus contemplate compositions that comprise one ormore BT compounds for use as antiseptics. An antiseptic is a substancethat kills or prevents the growth of microorganisms, and may betypically applied to living tissue, distinguishing the class fromdisinfectants, which are usually applied to inanimate objects (Goodmanand Gilman's “The Pharmacological Basis of Therapeutics”, SeventhEdition, Gilman et al., editors, 1985, Macmillan Publishing Co.,(hereafter, Goodman and Gilman”) pp. 959-960). Common examples ofantiseptics are ethyl alcohol and tincture of iodine. Germicides includeantiseptics that kill microbes such as microbial pathogens.

Certain embodiments described herein may contemplate compositions thatcomprise one or more BT compounds and one or more antibiotic compound(e.g., a synergizing antibiotic and/or an enhancing antibiotic asprovided herein). Antibiotics are known in the art and typicallycomprise a drug made from a compound produced by one species ofmicroorganism to kill another species of microorganism, or a syntheticproduct having an identical or similar chemical structure and mechanismof action, e.g., a drug that destroys microorganisms within or on thebody of a living organism, including such drug when applied topically.Among embodiments disclosed herein are those in which an antibiotic maybelong to one of the following classes: aminoglycosides, carbapenems,cephalosporins, fluoroquinolones, glycopeptide antibiotics, lincosamides(e.g., clindamycin), penicillinase-resistant penicillins, andaminopenicillins. Antibiotics thus may include, but need not be limitedto, oxacillin, piperacillin, cefuroxime, cefotaxime, cefepime, imipenem,aztreonam, streptomycin, tobramycin, tetracycline, minocycline,ciprofloxacin, levofloxacin, erythromycin, linezolid, phosphomycin,capreomycin, isoniazid, ansamycin, carbacephem, monobactam, nitrofuran,penicillin, quinolone, sulfonamide, Clofazimine, Dapsone, Capreomycin,Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide,Rifampicin, Rifampin, Rifabutin, Rifapentine, Streptomycin,Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Linezolid,Metronidazole, Mupirocin, Platensimycin, Quinupristin, Dalfopristin,Rifaximin, Thiamphenicol, Timidazole, aminoglycoside, beta-lactam,penicillin, cephalosporin, carbapenem, fluoroquinolone, ketolide,lincosamide, macrolide, oxazolidinone, stretogramin, sulphonamide,tetracycline, glycylcycline, methicillin, vancomycin, naficilin,gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin,amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin,clindamicin, gatifloxacin, aminopenicillin, and others known to the art.Compendia of these and other clinically useful antibiotics are availableand known to those familiar with the art (e.g., Washington UniversitySchool of Medicine, The Washington Manual of Medical Therapeutics(32^(nd) Ed.), 2007 Lippincott, Williams and Wilkins, Philadelphia, Pa.;Hauser, A L, Antibiotic Basics for Clinicians, 2007 Lippincott, Williamsand Wilkins, Philadelphia, Pa.).

An exemplary class of antibiotics for use with one or more BT compoundsin certain herein disclosed embodiments is the aminoglycoside class ofantibiotics, which are reviewed in Edson R S, Terrell C L. Theaminoglycosides. Mayo Clin Proc. 1999 May; 74(5):519-28. This class ofantibiotics inhibits bacterial growth by impairing bacterial proteinsynthesis, through binding and inactivation of bacterial ribosomalsubunits. In addition to such bacteriostatic properties, aminoglycosidesalso exhibit bacteriocidal effects through disruption of cell walls ingram-negative bacteria.

Aminoglycoside antibiotics include gentamicin, amikacin, streptomycin,and others, and are generally regarded as useful in the treatment ofgram-negative bacteria, mycobacteria and other microbial pathogens,although cases of resistant strains have been reported. Theaminoglycosides are not absorbed through the digestive tract and so arenot generally regarded as being amenable to oral formulations. Amikacin,for example, although often effective against gentamicin-resistantbacterial strains, is typically administered intravenously orintramuscularly, which can cause pain in the patient. Additionally,toxicities associated with aminoglycoside antibiotics such as amikacincan lead to kidney damage and/or irreversible hearing loss.

Despite these properties, certain embodiments disclosed hereincontemplate oral administration of a synergizing BT/antibioticcombination (e.g., where the antibiotic need not be limited to anaminoglycoside) for instance, for treatment of an epithelial tissuesurface at one or more locations along the oral cavity, gastrointestinaltract/alimentary canal. Also contemplated in certain other embodimentsmay be use of compositions and methods described herein asdisinfectants, which refers to preparations that kill, or block thegrowth of, microbes on an external surface of an inanimate object.

As also described elsewhere herein, a BT compound may be a compositionthat comprises bismuth or a bismuth salt and a thiol- (e.g., —SH, orsulfhydryl) containing compound, including those that are described(including their methods of preparation) in Domenico et al., 1997Antimicrob. Agent. Chemother. 41(8):1697-1703, Domenico et al., 2001Antimicob. Agent. Chemother. 45(5):1417-1421, and in U.S. RE37,793, U.S.Pat. No. 6,248,371, U.S. Pat. No. 6,086,921, and U.S. Pat. No.6,380,248; see also, e.g., U.S. Pat. No. 6,582,719. Certain embodimentsare not so limited, however, and may contemplate other BT compounds thatcomprise bismuth or a bismuth salt and a thiol-containing compound. Thethiol-containing compound may contain one, two, three, four, five, sixor more thiol (e.g., —SH) groups. In preferred embodiments the BTcompound comprises bismuth in association with the thiol-containingcompound via ionic bonding and/or as a coordination complex, while insome other embodiments bismuth may be associated with thethiol-containing compound via covalent bonding such as may be found inan organometallic compound. Certain contemplated embodiments, however,expressly exclude a BT compound that is an organometallic compound suchas a compound in which bismuth is found in covalent linkage to anorganic moiety.

Exemplary BT compounds are shown in Table 1:

TABLE 1 Exemplary BT Compounds*  1) CPD 1B-1 Bis-EDT (1:1) BiC₂H₄S₂  2)CPD 1B-2 Bis-EDT (1:1.5) BiC₃H₆S₃  3) CPD 1B-3 Bis-EDT (1:1.5) BiC₃H₆S₃ 4) CPD 1C Bis-EDT (1:1.5) BiC₃H₆S₃  5) CPD 2A Bis-Bal (1:1) BiC₃H₆S₂O 6) CPD 2B Bis-Bal (1:1.5) BiC_(4.5)H₉O_(1.5)S₃  7) CPD 3A Bis-Pyr(1:1.5) BiC_(7.5)H₆N_(1.5)O_(1.5)S_(1.5)  8) CPD 3B Bis-Pyr (1:3)BiC₁₅H₁₂N₃O₃S₃  9) CPD 4 Bis-Ery (1:1.5) BiC₆H₁₂O₃S₃ 10) CPD 5 Bis-Tol(1:1.5) BiC_(10.5)H₉S₃ 11) CPD 6 Bis-BDT (1:1.5) BiC₆H₁₂S₃ 12) CPD 7Bis-PDT (1:1.5) BiC_(4.5)H₉S₃ 13) CPD 8-1 Bis-Pyr/BDT (1:1/1) 14) CPD8-2 Bis-Pyr/BDT (1:1/0.5) 15) CPD 9 Bis-2hydroxy, propane thiol (1:3)16) CPD 10 Bis-Pyr/Bal (1:1/0.5) 17) CPD 11 Bis-Pyr/EDT (1:1/0.5) 18)CPD 12 Bis-Pyr/Tol (1:1/0.5) 19) CPD 13 Bis-Pyr/PDT (1:1/0.5) 20) CPD 14Bis-Pyr/Ery (1:1/0.5) 21) CPD 15 Bis-EDT/2hydroxy, propane thiol (1:1/1)*Shown are atomic ratios relative to a single bismuth atom, forcomparison, based on the stoichiometric ratios of the reactants used andthe known propensity of bismuth to form trivalent complexes with sulfurcontaining compounds. Atomic ratios as shown may not be accuratemolecular formulae for all species in a given preparation. The numbersin parenthesis are the ratios of bismuth to one (or more) thiol agents.(e.g. Bi:thiol1/thiol2) “CPD”, compound.

BT compounds for use in certain of the presently disclosed embodimentsmay be prepared according to established procedures (e.g., U.S.RE37,793, U.S. Pat. No. 6,248,371, U.S. Pat. No. 6,086,921, and U.S.Pat. No. 6,380,248; Domenico et al., 1997 Antimicrob. Agent. Chemother.41(8):1697-1703, Domenico et al., 2001 Antimicob. Agent. Chemother.45(5):1417-1421) and in certain other embodiments BT compounds may alsobe prepared according to methodologies described herein. Certainpreferred embodiments thus contemplate the herein described syntheticmethods for preparing BT compounds, and in particular for obtaining BTcompounds in substantially monodisperse microparticulate form, in whichan acidic aqueous bismuth solution that contains dissolved bismuth at aconcentration of at least 50 mM, at least 100 mM, at least 150 mM, atleast 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, atleast 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, atleast 800 mM, at least 900 mM or at least 1 M and that lacks ahydrophilic, polar or organic solubilizer is admixed with ethanol toobtain a first ethanolic solution, which is reacted with a secondethanolic solution comprising a thiol-containing compound to obtain areaction solution, wherein the thiol-containing compound is present inthe reaction solution at a molar ratio of from about 1:3 to about 3:1relative to the bismuth, under conditions and for a time sufficient forformation of a precipitate which comprises the microparticles comprisingthe BT compound (such as the conditions of concentration, solventstrength, temperature, pH, mixing and/or pressure, and the like, asdescribed herein and as will be appreciated by the skilled person basedon the present disclosure).

Accordingly, exemplary BTs include compound 1B-1, Bis-EDT(bismuth-1,2-ethane dithiol, reactants at 1:1); compound 1B-2, Bis-EDT(1:1.5); compound 1B-3, Bis-EDT (1:1.5); compound 1C, Bis-EDT (solubleBi preparation, 1:1.5); compound 2A, Bis-Bal (bismuth-Britishanti-Lewisite (bismuth-dimercaprol, bismuth-2,3-dimercaptopropanol),1:1); compound 2B, Bis-Bal (1:1.5); compound 3A Bis-Pyr(bismuth-pyrithione, 1:1.5); compound 3B Bis-Pyr (1:3); compound 4,Bis-Ery (bismuth-dithioerythritol, 1:1.5); compound 5, Bis-Tol(bismuth-3,4-dimercaptotoluene, 1:1.5); compound 6, Bis-BDT(bismuth-2,3-butanedithiol, 1:1.5); compound 7, Bis-PDT(bismuth-1,3-propanedithiol, 1:1.5); compound 8-1 Bis-Pyr/BDT (1:1/1);compound 8-2, Bis-Pyr/BDT (1:1/0.5); compound 9, Bis-2-hydroxy, propanethiol (bismuth-1-mercapto-2-propanol, 1:3); compound 10, Bis-Pyr/Bal(1:1/0.5); compound 11, Bis-Pyr/EDT (1:1/0.5); compound 12 Bis-Pyr/Tol(1:1/0.5); compound 13, Bis-Pyr/PDT (1:1/0.5); compound 14 Bis-Pyr/Ery(1:1/0.5); compound 15, Bis-EDT/2-hydroxy, propane thiol (1:1/1) (see,e.g., Table 1).

Without wishing to be bound by theory, it is believed that the presentlydisclosed methods of preparing a BT compound, which in certain preferredembodiments may comprise preparing or obtaining an acidic aqueous liquidsolution that comprises bismuth such as an aqueous nitric acid solutioncomprising bismuth nitrate, may desirably yield compositions comprisingBT compounds where such compositions have one or more desirableproperties, including ease of large-scale production, improved productpurity, uniformity or consistency (including uniformity in particlesize), or other properties useful in the preparation and/oradministration of the present topical formulations.

In particular embodiments it has been discovered that BT compositions,prepared according to the methods described herein for the first time,exhibit an advantageous degree of homogeneity with respect to theiroccurrence as a substantially monodisperse suspension of microparticleseach having a volumetric mean diameter (VMD) according to certainpresently preferred embodiments of from about 0.4 μm to about 5 μm.Measures of particle size can be referred to as volumetric mean diameter(VMD), mass median diameter (MMD), or mass median aerodynamic diameter(MMAD). These measurements may be made, for example, by impaction (MMDand MMAD) or by laser (VMD) characterization. For liquid particles, VMD,MMD and MMAD may be the same if environmental conditions are maintained,e.g., standard humidity. However, if humidity is not maintained, MMD andMMAD determinations will be smaller than VMD due to dehydration duringimpactor measurements. For the purposes of this description, VMD, MMDand MMAD measurements are considered to be under standard conditionssuch that descriptions of VMD, MMD and MMAD will be comparable.Similarly, dry powder particle size determinations in MMD, and MMAD arealso considered comparable.

As described herein, preferred embodiments relate to a substantiallymonodisperse suspension of BT-containing microparticles. Generation of adefined BT particle size with limited geometric standard deviation (GSD)may, for instance, optimize BT deposition, accessibility to desiredtarget sites in or on a natural surface, and/or tolerability by asubject to whom the BT microparticles are administered. Narrow GSDlimits the number of particles outside the desired VMD or MMAD sizerange.

In one embodiment, a liquid or aerosol suspension of microparticlescontaining one or more BT compounds disclosed herein is provided havinga VMD from about 0.5 microns to about 5 microns. In another embodiment,a liquid or aerosol suspension having a VMD or MMAD from about 0.7microns to about 4.0 microns is provided. In another embodiment, aliquid or aerosol suspension having aVMD or MMAD from about 1.0 micronto about 3.0 microns is provided. In certain other preferred embodimentsthere is provided a liquid suspension comprising one or a plurality ofBT compound particles of from about 0.1 to about 5.0 microns VMD, or offrom about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6,about 0.7, about 0.8 or about 0.9 microns to about 1.0, about 1.5, about2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0,about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 or about 8.0microns, the particle comprising a BT compound prepared as describedherein.

Accordingly and in certain preferred embodiments, a BT preparationdescribed for the first time herein which is “substantially”monodisperse, for example, a BT composition that comprises a BT compoundin microparticulate form wherein “substantially” all of themicroparticles have a volumetric mean diameter (VMD) within a specifiedrange (e.g., from about 0.4 μm to about 5 μm), includes thosecompositions in which at least 80%, 85%, 90%, 91%, 92%, 93%, or 94%,more preferably at least 95%, 96%, 97%, 98%, 99% or more of theparticles have a VMD that is within the recited size range.

These and related properties of BT compositions prepared according tothe herein described synthetic methods offer unprecedented advantagesover previously described BTs, including lower cost and ease ofproduction, and uniformity within the composition that may permit itscharacterization in a manner that facilitates regulatory complianceaccording to one or more of pharmaceutical, formulary and cosmeceuticalstandards.

Additionally or alternatively, the herein described substantiallymonodisperse BT microparticles may advantageously be produced withoutthe need for micronization, i.e., without the expensive andlabor-intensive milling or supercritical fluid processing or otherequipment and procedures that are typically used to generatemicroparticles (e.g., Martin et al. 2008 Adv. Drug Deliv. Rev.60(3):339; Moribe et al., 2008 Adv. Drug Deliv. Rev. 60(3):328; Cape etal., 2008 Pharm. Res. 25(9):1967; Rasenack et al. 2004 Pharm. Dev.Technol. 9(1):1-13). Hence, the present embodiments offer beneficialeffects of substantially uniform microparticulate preparations,including without limitation enhanced and substantially uniformsolubilization properties, suitability for desired administration formssuch as oral, inhaled or dermatological/skin wound topical forms,increased bioavailability and other beneficial properties.

The BT compound microparticulate suspension can be administered asaqueous formulations, as suspensions or solutions in aqueous as well asorganic solvents including halogenated hydrocarbon propellants, as drypowders, or in other forms as elaborated below, including preparationsthat contain wetting agents, surfactants, mineral oil or otheringredients or additives as may be known to those familiar withformulary, for example, to maintain individual microparticles insuspension. Aqueous formulations may be aerosolized by liquid nebulizersemploying, for instance, either hydraulic or ultrasonic atomization.Propellant-based systems may use suitable pressurized dispensers. Drypowders may use dry powder dispersion devices, which are capable ofdispersing the BT-containing microparticles effectively. A desiredparticle size and distribution may be obtained by choosing anappropriate device.

As also noted above, also provided herein according to certainembodiments is a method for preparing a bismuth-thiol (BT) compositionthat comprises a plurality of microparticles that comprise a BTcompound, substantially all of such microparticles having a volumetricmean diameter (VMD) of from about 0.1 to about 8 microns, and in certainpreferred embodiments from about 0.4 microns to about 5 microns.

In general terms, the method comprises the steps of (a) admixing, underconditions and for a time sufficient to obtain a solution that issubstantially free of a solid precipitate, (i) an acidic aqueoussolution that comprises a bismuth salt comprising bismuth at aconcentration of at least 50 mM and that lacks a hydrophilic, polar ororganic solubilizer, with (ii) ethanol in an amount sufficient to obtainan admixture that comprises at least about 5%, 10%, 15%, 20%, 25% or30%, and preferably about 25% ethanol by volume; and (b) adding to theadmixture of (a) an ethanolic solution comprising a thiol-containingcompound to obtain a reaction solution, wherein the thiol-containingcompound is present in the reaction solution at a molar ratio of fromabout 1:3 to about 3:1 relative to the bismuth, under conditions and fora time sufficient for formation of a precipitate which comprises the BTcompound.

In certain preferred embodiments the bismuth salt may be Bi(NO₃)₃, butit will be appreciated according to the present disclosure that bismuthmay also be provided in other forms. In certain embodiments the bismuthconcentration in the acidic aqueous solution may be at least 100 mM, atleast 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, atleast 350 mM, at least 400 mM, at least 500 mM, at least 600 mM, atleast 700 mM, at least 800 mM, at least 900 mM or at least 1 M. Incertain embodiments the acidic aqueous solution comprises at least 5%,10%, 15%, 20%, 22% or 22.5% bismuth by weight. The acidic aqueoussolution may in certain preferred embodiments comprise at least 5% ormore nitric acid by weight, and in certain other embodiments the acidicaqueous solution may comprise at least 0.5%, at least 1%, at least 1.5%,at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, atleast 4.5% or at least 5% nitric acid by weight.

The thiol-containing compound may be any thiol-containing compound asdescribed herein, and in certain embodiments may comprise one or more of1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol,1,3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-propanol,dithioerythritol and dithiothreitol. Other exemplary thiol-containingcompounds include alpha-lipoic acid, methanethiol (CH₃SH [m-mercaptan]),ethanethiol (C₂H₅SH [e-mercaptan]), 1-propanethiol (C₃H₇SH [n-Pmercaptan]), 2-Propanethiol (CH₃CH(SH)CH₃ [2C₃ mercaptan]), butanethiol(C₄H₉SH ([n-butyl mercaptan]), tert-butyl mercaptan (C(CH₃)₃SH [t-butylmercaptan]), pentanethiols (C₅H₁₁SH [pentyl mercaptan]), coenzyme A,lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol,dithiothreitol, dithioerythritol, 2-mercaptoindole, transglutaminase andany of the following thiol compounds available from Sigma-Aldrich (St.Louis, Mo.): (11-mercaptoundecyl)hexa(ethylene glycol),(11-mercaptoundecyl)tetra(ethylene glycol),(11-mercaptoundecyl)tetra(ethylene glycol) functionalized goldnanoparticles, 1,1′,4′,1″-terphenyl-4-thiol, 1,11-undecanedithiol,1,16-hexadecanedithiol, 1,2-ethanedithiol technical grade,1,3-propanedithiol, 1,4-benzenedimethanethiol, 1,4-butanedithiol,1,4-butanedithiol diacetate, 1,5-pentanedithiol, 1,6-hexanedithiol,1,8-octanedithiol, 1,9-nonanedithiol, adamantanethiol, 1-butanethiol,1-decanethiol, 1-dodecanethiol, 1-heptanethiol, 1-heptanethiol purum,1-hexadecanethiol, 1-hexanethiol, 1-mercapto-(triethylene glycol),1-mercapto-(triethylene glycol) methyl ether functionalized goldnanoparticles, 1-mercapto-2-propanol, 1-nonanethiol, 1-octadecanethiol,1-octanethiol, 1-octanethiol, 1-pentadecanethiol, 1-pentanethiol,1-propanethiol, 1-tetradecanethiol, 1-tetradecanethiol purum,1-undecanethiol, 11-(1H-pyrrol-1-yl)undecane-1-thiol,11-amino-1-undecanethiol hydrochloride, 11-bromo-1-undecanethiol,11-mercapto-1-undecanol, 11-mercapto-1-undecanol, 11-mercaptoundecanoicacid, 11-mercaptoundecanoic acid, 11-mercaptoundecyl trifluoroacetate,11-mercaptoundecylphosphoric acid, 12-mercaptododecanoic acid,12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid,16-mercaptohexadecanoic acid, 16-mercaptohexadecanoic acid,1H,1H,2H,2H-perfluorodecanethiol, 2,2′-(ethylenedioxy)diethanethiol,2,3-butanedithiol, 2-butanethiol, 2-ethylhexanethiol,2-methyl-1-propanethiol, 2-methyl-2-propanethiol, 2-phenylethanethiol,3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanethiol purum,3-(dimethoxymethylsilyl)-1-propanethiol, 3-chloro-1-propanethiol,3-mercapto-1-propanol, 3-mercapto-2-butanol,3-mercapto-N-nonylpropionamide, 3-mercaptopropionic acid,3-mercaptopropyl-functionalized silica gel, 3-methyl-1-butanethiol,4,4′-bis(mercaptomethyl)biphenyl, 4,4′-dimercaptostilbene,4-(6-mercaptohexyloxyl)benzyl alcohol, 4-cyano-1-butanethiol,4-mercapto-1-butanol, 6-(ferrocenyl)hexanethiol, 6-mercapto-1-hexanol,6-mercaptohexanoic acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid,9-mercapto-1-nonanol, biphenyl-4,4′-dithiol, butyl 3-mercaptopropionate,copper(I) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol,decanethiol functionalized silver nanoparticles, dodecanethiolfunctionalized gold nanoparticles, dodecanethiol functionalized silvernanoparticles, hexa(ethylene glycol)mono-11-(acetylthio)undecyl ether,mercaptosuccinic acid, methyl 3-mercaptopropionate, heptanoic hydrazidebisphenol A substituted5-(Hydroxymethyl)-1,3-bis(6-mercaptohexyloxy)benzene sold under thetrademark NANO TETHER BPA-HH™ (Sigma-Aldrich, St. Louis, Mo.),octadecanethiol reagents sold under the trademark NANOTHINKS™ 18(Sigma-Aldrich), octanethiol reagents sold under the trademarkNANOTHINKS™ 8 (Sigma-Aldrich), 11-mercaptoundecanoic acid reagents soldunder the trademark NANOTHINKS™ ACID11 (Sigma-Aldrich),16-mercaptorhexadecanoic acid reagents sold under the trademarkNANOTHINKS™ ACID16 (Sigma-Aldrich), 11-mercapto-1-undecanol reagentssold under the trademark NANOTHINKS™ ALCO11 (Sigma-Aldrich),1,8-octanedithiol sold under the trademark NANOTHINKS™ THIO8(Sigma-Aldrich), octanethiol functionalized gold nanoparticles, PEGdithiol average M_(n) 8,000, PEG dithiol average mol wt 1,500, PEGdithiol average mol wt 3,400, S-(11-bromoundecyl)thioacetate,S-(4-cyanobutyl)thioacetate, thiophenol, triethylene glycolmono-11-mercaptoundecyl ether, trimethylolpropanetris(3-mercaptopropionate),[11-(methylcarbonylthio)undecyl]tetra(ethylene glycol),m-carborane-9-thiol, p-terphenyl-4,4″-dithiol, tert-dodecylmercaptan,and tert-nonyl mercaptan.

Exemplary reaction conditions, including temperature, pH, reaction time,the use of stirring or agitation to dissolve solutes and procedures forcollecting and washing precipitates, are described herein and employtechniques generally known in the art.

Unlike previously described methodologies for producing BT compounds,according to the present methods for preparing BT, BT products areprovided as microparticulate suspensions having substantially allmicroparticles with VMD from about 0.4 to about 5 microns in certainpreferred embodiments, and generally from about 0.1 microns to about 8microns according to certain other embodiments. Further unlike previousapproaches, according to the instant embodiments bismuth is provided inan acidic aqueous solution that comprises a bismuth salt at aconcentration of from at least about 50 mM to about 1 M, and nitric acidin an amount from at least about 0.5% to about 5% (w/w), and preferablyless than 5% (weight/weight), and that lacks a hydrophilic, polar ororganic solubilizer.

In this regard the present methods offer surprising and unexpectedadvantages in view of generally accepted art teachings that bismuth isnot water soluble at 50 μM (e.g., U.S. RE37793), that bismuth isunstable in water (e.g., Kuvshinova et al., 2009 Russ. J Inorg. Chem.54(11):1816), and that bismuth is unstable even in nitric acid solutionsunless a hydrophilic, polar or organic solubilizer is present. Forexample, in all of the definitive descriptions of BT preparationmethodologies (e.g., Domenico et al., 1997 Antimicrob. Agents.Chemother. 41:1697; U.S. Pat. No. 6,380,248; U.S. RE37793; U.S. Pat. No.6,248,371), the hydrophilic solubilizing agent propylene glycol isrequired to dissolve bismuth nitrate, and the bismuth concentration ofsolutions prepared for reaction with thiols is well below 15 mM, therebylimiting the available production modalities for BT compounds.

By contrast, according to the present disclosure there is no requirementfor a hydrophilic, polar or organic solubilizer in order dissolvebismuth, yet higher concentrations are surprisingly achieved.Hydrophilic, polar or organic solubilizers include propylene glycol (PG)and ethylene glycol (EG) and may also include any of a large number ofknown solubility enhancers, including polar solvents such as dioxane anddimethylsulfoxide (DMSO), polyols (including, e.g., PG and EG and alsoincluding polyethylene glycol (PEG), polypropyleneglycol (PPG),pentaerythritol and others), polyhydric alchohols such as glycerol andmannitol, and other agents. Other water-miscible organic of highpolarity include dimethylsulfoxide (DMSO), dimethylformamide (DMF) andNMP (N-methyl-2-pyrrolidone).

Thus, it will be appreciated by those familiar with the art thatsolvents, including those commonly used as hydrophilic, polar or organicsolubilizers as provided herein, may be selected, for instance, based onthe solvent polarity/polarizability (SPP) scale value using the systemof Catalan et al. (e.g., 1995 Liebigs Ann. 241; see also Catalan, 2001In: Handbook of Solvents, Wypych (Ed.), Andrew Publ., NY, and referencescited therein), according to which, for example, water has a SPP valueof 0.962, toluene a SPP value of 0.655, and 2-propanol a SPP value of0.848. Methods for determining the SPP value of a solvent based onultraviolet measurements of the2-N,N-dimethyl-7-nitrofluorene/2-fluoro-7-nitrofluorene probe/homomorphpair have been described (Catalan et al., 1995).

Solvents with desired SPP values (whether as pure single-componentsolvents or as solvent mixtures of two, three, four or more solvents;for solvent miscibility see, e.g., Godfrey 1972 Chem. Technol. 2:359)based on the solubility properties of a particular BT composition can bereadily identified by those having familiarity with the art in view ofthe instant disclosure, although as noted above, according to certainpreferred embodiments regarding the herein described synthetic methodsteps, no hydrophilic, polar or organic solubilizer is required in orderdissolve bismuth.

Solubility parameters may also include the interaction parameter C,Hildebrand solubility parameter d, or partial (Hansen) solubilityparameters: δp, δh and δd, describing the solvent's polarity, hydrogenbonding potential and dispersion force interaction potential,respectively. In certain embodiments, the highest value for a solubilityparameter that describes a solvent or co-solvent system in which thebismuth salt comprising bismuth will dissolve may provide a limitationfor the aqueous solution that comprises the bismuth salt, for instance,according to the presently described method for preparing amicroparticulate BT composition. For example, higher δh values will havea greater hydrogen bonding ability and would therefore have a greateraffinity for solvent molecules such as water. A higher value of maximumobserved δh for a solvent may therefore be preferred for situationswhere a more hydrophilic environment is desired.

By way of non-limiting example, BisEDT having the structure shown belowin formula I may be prepared according to the following reaction scheme:

Briefly, and as a non-limiting illustrative example, to an excess (11.4L) of 5% aqueous HNO₃ at room temperature may be slowly added 0.331 L(about 0.575 moles) of an aqueous acidic bismuth solution such as aBi(NO₃)₃ solution (e.g., 43% Bi(NO₃)₃ (w/w), 5% nitric acid (w/w), 52%water (w/w), available from Shepherd Chemical Co., Cincinnati, Ohio)with stirring, followed by slow addition of absolute ethanol (4 L). Anethanolic solution (1.56 L) of a thiol compound such as 1,2-ethanedithio[˜0.55 M] may be separately prepared by adding, to 1.5 L of absoluteethanol, 72.19 mL (0.863 moles) of 1,2-ethanedithiol using a 60 mLsyringe, and then stirring for five minutes. 1,2-ethanedithiol (CAS540-63-6) and other thiol compounds are available from, e.g.,Sigma-Aldrich, St. Louis, Mo. The ethanolic solution of the thiolcompound may then be slowly added to the aqueous Bi(NO₃)₃/HNO₃ solutionwith stirring overnight to form a reaction solution. Thethiol-containing compound may be present in the reaction solution,according to certain preferred embodiments, at a molar ratio of fromabout 1:3 to about 3:1 relative to the bismuth. The formed product isallowed to settle as a precipitate comprising microparticles asdescribed herein, which is then collected by filtration and washedsequentially with ethanol, water and acetone to obtain BisEDT as ayellow amorphous powdered solid. The crude product may be redissolved inabsolute ethanol with stirring, then filtered and washed sequentiallywith ethanol several times followed by acetone several times. The washedpowder may be triturated in 1M NaOH (500 mL), filtered and washedsequentially with water, ethanol and acetone to afford purifiedmicroparticulate BisEDT.

According to non-limiting theory, bismuth inhibits the ability ofbacteria to produce extracellular polymeric substances (EPS) such asbacterial exopolysaccharides, and this inhibition leads to impairedbiofilm formation. Bacteria are believed to employ the glue-like EPS forbiofilm cohesion. Depending on the nature of an infection, biofilmformation and elaboration of EPS may contribute to bacterialpathogenicity such as interference with wound healing. However, bismuthalone is not therapeutically useful as an intervention agent, and isinstead typically administered as part of a complex such as a BT.Bismuth-thiols (BTs) are thus a family of compositions that includescompounds that result from the chelation of bismuth with a thiolcompound, and that exhibit dramatic improvement in the antimicrobialtherapeutic efficacy of bismuth. BTs exhibit remarkable anti-infective,anti-biofilm, and immunomodulatory effects. Bismuth thiols are effectiveagainst a broad-spectrum of microorganisms, and are typically notaffected by antibiotic-resistance. BTs prevent biofilm formation atremarkably low (sub-inhibitory) concentrations, prevent many pathogeniccharacteristics of common wound pathogens at those same sub-inhibitorylevels, can prevent septic shock in animal models, and may besynergistic with many antibiotics.

As described herein, such synergy in the antibacterial effects of one ormore specified BT when combined with one or more specified antibioticcompound is not readily predictable based on profiles of separateantibiotic and BT effects against a particular bacterial type, butsurprisingly may result from selection of particular BT-antibioticcombinations in view of the specific bacterial population, includingidentification of whether gram-negative or gram-positive (or both)bacteria are present. For instance, as disclosed herein, antibioticsthat synergize with certain BTs may include one or more of amikacin,ampicillin, aztreonam, cefazolin, cefepime, chloramphenicol,ciprofloxacin, clindamycin (or other lincosamide antibiotics),daptomycin (sold under the trademark CUBICIN®), doxycycline,gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (sold underthe trademark ZYVOX®), minocycline, nafcilin, paromomycin, rifampin,sulphamethoxazole, tetracycline, tobramycin and vancomycin. In vitrostudies showed, for example, that MRSA, which was poorly or not at allsusceptible to gentamicin, cefazolin, cefepime, suphamethoxazole,imipenim or levofloxacin individually, exhibited marked sensitivity toany one of these antibiotics if exposed to the antibiotic in thepresence of the BT compound BisEDT. Certain embodiments contemplatedherein thus expressly contemplate compositions and/or methods in whichmay be included the combination of a BT compound and one or moreantibiotics selected from amikacin, ampicillin, cefazolin, cefepime,chloramphenicol, ciprofloxacin, clindamycin (or another lincosamideantibiotic), daptomycin (sold under the trademark CUBICIN®),doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid(sold under the trademark ZYVOX®), minocycline, nafcilin, paromomycin,rifampin, sulphamethoxazole, tobramycin and vancomycin, whilst certainother embodiments contemplated herein contemplate compositions and/ormethods in which may be included the combination of a BT compound andone or more antibiotics from which expressly excluded may be one or moreantibiotic selected from amikacin, ampicillin, cefazolin, cefepime,chloramphenicol, ciprofloxacin, clindamycin (or other lincosamides),daptomycin (sold under the trademark CUBICIN®), doxycycline,gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (sold underthe trademark ZYVOX®), minocycline, nafcilin, paromomycin, rifampin,sulphamethoxazole, tobramycin and vancomycin. It is noted in thiscontext that gentamicin and tobramycin belong to the aminoglycosideclass of antibiotics. Also expressly excluded from certain contemplatedembodiments are certain compositions and methods described in Domenicoet al., 2001 Agents Chemother. 45:1417-1421; Domenico et al., 2000Infect. Med. 17:123-127; Domenico et al., 2003 Res. Adv. In Antimicrob.Agents & Chemother. 3:79-85; Domenico et al., 1997 Antimicrob. AgentsChemother. 41(8):1697-1703; Domenico et al., 1999 Infect. Immun.67:664-669: Huang et al. 1999 J Antimicrob. Chemother. 44:601-605;Veloira et al., 2003 J Antimicrob. Chemother. 52:915-919; Wu et al.,2002 Am J Respir Cell Mol Biol. 26:731-738; Halwani et al., 2008 Int. JPharmaceut. 358:278; Halwani et al., 2009 Int. J. Pharmaceut.373:141-146; where it will be noted that none of these publicationsteach or suggest the mondisperse microparticulate BT compositions thatare disclosed herein.

Accordingly and as described herein, in certain preferred embodimentsthere are provided compositions and methods for treating a subject witha composition that comprises the herein described microparticulate BTand that optionally and in certain other embodiments also comprises asynergizing and/or an enhancing antibiotic. Persons familiar with therelevant art will, based on the present disclosure, recognizeappropriate clinical contexts and situations in which such treatment maybe desired, criteria for which are established in the medical arts,including inter alia, e.g., surgical, military surgical, dermatological,trauma medicine, gerontological, cardiovascular, metabolic diseases(e.g., diabetes, obesity, etc.), infection and inflammation (includingin the epithelial linings of the respiratory tract or thegastrointestinal tract, or other epithelial tissue surfaces such as inglandular tissues), and other relevant medical specialties andsubspecialties.

It will therefore be appreciated that, in certain embodiments asdisclosed herein and known in the art, promoting skin tissue repair (orother tissue repair, such as epithelial tissue, bone, joint, muscletendon, or ligament repair) is contemplated. In certain embodiments,promoting skin tissue or other epithelial tissue repair may comprisestimulating or disinhibiting one or more cellular wound repairactivities selected from (i) epithelial cell (e.g., keratinocyte) ordermal fibroblast migration, (ii) epithelial cell (e.g., keratinocyte)or dermal fibroblast growth, (iii) downregulation of epithelial cell(e.g., keratinocyte) or dermal fibroblast collagenase, gelatinase ormatrix metalloproteinase activity, (iv) dermal fibroblast extracellularmatrix protein deposition, and (v) induction or potentiation of dermalangiogenesis. Methodologies for identifying and characterizing suchcellular wound repair activities have been described such that theeffects of the herein disclosed wound tissue repair-promoting compounds,such as compositions comprising BT agents as described herein, on theseand related activities can be determined readily and without undueexperimentation based on the present disclosure. For example, disclosedherein are compositions and methods that relate to art accepted modelsfor wound repair based on keratinocyte wound closure following a scratchwound.

Preferred compositions for treating a microbial infection on or in anatural surface for use according to the embodiments described herein,may include in certain embodiments compositions that comprisebismuth-thiol (BT) compounds as described herein, and which may incertain distinct but related embodiments also include other compoundsthat are known in the art such as one or more antibiotic compounds asdescribed herein. BT compounds and methods for making them are disclosedherein and are also disclosed, for example, in Domenico et al. (1997Antimicrob. Agent. Chemother. 41(8):1697-1703; 2001 Antimicrob. Agent.Chemother. 45(5)1417-1421) and in U.S. RE37,793, U.S. Pat. No.6,248,371, U.S. Pat. No. 6,086,921, and U.S. Pat. No. 6,380,248. As alsonoted above, certain preferred BT compounds are those that containbismuth or a bismuth salt ionically bonded to, or in a coordinationcomplex with, a thiol-containing compound, such as a composition thatcomprises bismuth chelated to the thiol-containing compound, and certainother preferred BT compounds are those that contain bismuth or a bismuthsalt in covalent bond linkage to the thiol-containing compound. Alsopreferred are substantially monodisperse microparticulate BTcompositions as described herein. Neither from previous efforts to treatbacterial infections, nor from previous characterization in othercontexts of any compounds described herein for the first time as havinguse in compositions and methods for promoting the herein describedtreatment of natural surfaces, could it be predicted that the presentmethods of using such compounds would have the herein describedbeneficial effects.

According to preferred embodiments there are thus provided methods fortreating a natural surface, comprising administering to the surface atleast one microparticulate BT compound as described herein. In certainembodiments the method further comprises administering, simultaneouslyor sequentially and in either order, at least one antibiotic compound,which in certain preferred embodiments may be a synergizing antibioticas described herein, and which in certain other preferred embodimentsmay be an enhancing antibiotic as described herein. The antibioticcompound may be an aminoglycoside antibiotic, a carbapenem antibiotic, acephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptidesantibiotic, a lincosamide antibiotic, a penicillinase-resistantpenicillin antibiotic, or an aminopenicillin antibiotic. Clinicallyuseful antibiotics are discussed elsewhere herein and are also describedin, e.g., Washington University School of Medicine, The WashingtonManual of Medical Therapeutics (32^(nd) Ed.), 2007 Lippincott, Williamsand Wilkins, Philadelphia, Pa.; and in Hauser, A L, Antibiotic Basicsfor Clinicians, 2007 Lippincott, Williams and Wilkins, Philadelphia, Pa.

As described herein, certain embodiments derive from the unpredictablediscovery that for a bacterial infection that comprises gram positivebacteria, a preferred therapeutically effective formulation may comprisea BT compound (e.g., BisEDT, bismuth:1,2-ethanedithiol; BisPyr,bismuth:pyrithione; BisEDT/Pyr, bismuth:1,2-ethanedithiol/pyrithione)and rifamycin, or a BT compound and daptomycin (sold under the trademarkCUBICIN®, Cubist Pharmaceuticals, Lexington, Mass.), or a BT compoundand linezolid (sold under the trademark ZYVOX®, Pfizer, Inc., NY, NY),or a BT compound (e.g., BisEDT, bismuth:1,2-ethanedithiol; BisPyr,bismuth:pyrithione; BisEDT/Pyr, bismuth:1,2-ethanedithiol/pyrithione)and one or more of ampicillin, cefazolin, cefepime, chloramphenicol,clindamycin (or another lincosamide antibiotic), daptomycin (CUBICIN®),doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid(ZYVOX®), nafcilin, paromomycin, rifampin, sulphamethoxazole, tobramycinand vancomycin.

As also described herein, certain embodiments derive from theunpredictable discovery that for a bacterial infection that comprisesgram negative bacteria, a preferred therapeutically effectiveformulation may comprise a BT compound and amikacin. Certain relatedembodiments contemplate treatment of an infection comprising gramnegative bacteria with a BT compound and another antibiotic, such asanother aminoglycoside antibiotic, which in certain embodiments is notgentamicin or tobramycin. Accordingly and in view of these embodiments,other related embodiments contemplate identifying one or more bacterialpopulations or subpopulations in or on a natural surface by the wellknown criterion of being gram positive or gram negative, according tomethodologies that are familiar to those skilled in the medicalmicrobiology art, as a step for selecting appropriate antibioticcompound(s) to include in a formulation to be administered according tothe present methods.

The presently described compositions and methods may find use in thetreatment of microbes (e.g., bacteria, viruses, yeast, molds and otherfungi, microbial parasites, etc.) in a wide variety of contexts,typically by application or administration of the herein describedcompounds (e.g., one or more microparticulate BTs alone or incombination with one or more synergizing and/or enhancing antibiotics asdisclosed herein) to a microbial site such as a microbial presence on orin a natural surface. Such natural surfaces include but are not limitedto mammalian tissues (e.g., epithelia including skin, scalp,gastrointestinal tract lining, buccal cavity, etc.; endothelia, cell andtissue membranes such as peritoneal membrane, pericardial membrane,pleural membrane, periosteal membrane, meningeal membranes, sarcolemalmembranes, and the like; cornea, sclera, mucous membranes, etc.; andother mammalian tissues such as teeth, bone, joint, tendon, ligament,muscle, heart, lung, kidney, liver, spleen, gall bladder, pancreas,bladder, nerve, etc.).

The microparticulate antimicrobial agents described herein may be usedto suppress microbial growth, reduce microbial infestation, reducebiofilm, prevent conversion of bacteria to biofilm, prevent or inhibitmicrobial infection and any other use described herein. These agents arealso useful for a number of antiviral purposes, including prevention orinhibition of viral infection by herpes family viruses such ascytomegalovirus, herpes simplex virus Type 1, and herpes simplex virusType 2, and/or infection by other viruses. In this regard, the agentsare useful for the prevention or inhibition of viral infection by avariety of viruses, such as, single stranded RNA viruses, singlestranded DNA viruses, Rous sarcoma virus (RSV), hepatitis A virus,hepatitis B virus (HBV), Hepatitis C(HCV), Influenza viruses, west nilevirus (WNV), Epstein-Barr virus (EBV), eastern equine encephalitis virus(EEEV), severe acute respiratory virus (SARS), human immunodeficiencyvirus (HIV), human papilloma virus (HPV), and human T cell lymphomavirus (HTLV).

Other internal and external pharmaceutical uses of the herein describedantimicrobial agents include, but are not limited to, treatment orprevention of bacterial infection, of tuberculosis, of fungal infectionssuch as yeast and mold infections (for example, Candida (e.g., Candidaalbicans, Candida glabrata, C. parapsilosis, C. tropicalis, and C.dubliniensis) or Cryptococcus or other fungi), of Helicobacter pyloriinfection, and of peptic ulcer disease. In one embodiment, the agent isused at a dosage not generally lethal to bacteria but which isnonetheless sufficient to reduce protective polysaccharide coatings thatwould otherwise resist natural immune response. This technique is thusbelieved to aid immune system-mediated eradication of bacterialinfection without harming human symbiotic microorganisms (e.g., normalintestinal flora and the like) to the extent that may be the case withantibiotics.

By way of illustration and not limitation, certain contemplatedembodiments are now described.

In certain embodiments, a microparticulate BT compound described herein(or composition comprising the microparticulate BT compound) may becombined with at least one or more anti-biofilm agents for controllingbiofilm development, disrupting a biofilm, or reducing the amount ofbiofilm. As understood in the art, interspecies quorum sensing isrelated to biofilm formation. Certain agents that increaseLuxS-dependent pathway or interspecies quorum sensing signal (see, e.g.,U.S. Pat. No. 7,427,408) contribute to controlling development and/orproliferation of a biofilm. Exemplary agents include, by way of example,N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) blocking compounds andN-butyryl-L-homoserine lactone (BHL) analogs, either in combination orseparately (see, e.g., U.S. Pat. No. 6,455,031). An oral hygienecomposition comprising a microparticulate BT compound and at least oneanti-biofilm agent can be delivered locally for disruption andinhibition of bacterial biofilm and for treatment of periodontal disease(see, e.g., U.S. Pat. No. 6,726,898).

Compositions Comprising Microparticulate Bismuth-Thiols and Uses forOral Hygiene and for Treating Inflammation and Infection of the Mouth.

In another embodiment, compositions comprising microparticulate BTcompounds are formulated for oral use and may be used in methods forpreventing or reducing microbial growth in the mouth and for preventingand/or treating microbial infections and inflammation of the oralcavity. These compositions are therefore useful for preventing ortreating (i.e., reducing or inhibiting development of, reducing thelikelihood of occurrence or recurrence of) dental plaque, halitosis,periodontal disease, gingivitis, and other infections of the mouth. Theoral compositions comprising microparticulate BT compound may also beuseful for preventing and/or controlling (i.e., slowing, retarding,inhibiting) biofilm development, disrupting a biofilm, or reducing theamount of biofilm present on an oral surface, particularly a tooth orgums.

Trapped food particles, poor oral hygiene and poor oral health, andimproper cleaning of dentures can promote microbial growth betweenteeth, around the gums, and on the tongue. Continued microbial growthand the presence of dental caries may result in halitosis, dental plaque(i.e., a biofilm formed by colonization of microorganisms), gingivitis,and inflammation. In the absence of proper oral care (e.g., toothbrushing, flossing), more serious infections, such as periodontaldisease and infections of the jaw, may ensue.

Good oral hygiene is important not only for oral health, but forprevention of several chronic conditions. Controlling bacterial growthin the mouth may help lower risk of heart disease, preserve memory, andreduce the risk of infection and inflammation in other areas of thebody. People with diabetes are at greater risk for developing severe gumproblems, and reducing the risk of gingivitis by maintaining good oralhealth may help control blood sugar. Pregnant women may be more likelyto experience gingivitis, and some research suggests a relationshipbetween gum disease in pregnant women and delivery of preterm,low-birth-weight infants.

Bacteria are the primary etiologic agents in periodontal disease. Morethan 500 bacterial strains may be found in dental plaque (Kroes et al.,Proc. Natl. Acad. Sci. USA 96:14547-52 (1999)). Bacteria have evolved tosurvive in the environment of the tooth surface, gingival epithelium,and oral cavity as biofilms, which contributes to the difficulty intreating periodontitis. Bactericidal agents as well as antibiotics thatare currently used to treat such infections often do not kill all ofoffending organisms. Use of an agent that is ineffective against certainbacteria species may result in proliferation of resistant bacterialspecies. Moreover, these agents may cause unpleasant side effects, suchallergic reactions, inflammation, and tooth discoloration.

Dental bacterial plaque is a biofilm that adheres tenaciously to toothsurfaces, restorations, and prosthetic appliances. The primary means tocontrol biofilms in the mouth is through mechanical cleaning (i.e.,tootbrushing, flossing, etc.). Within the first two days after which nosuch cleaning has been undertaken, the tooth's surface is colonizedpredominantly by gram-positive facultative cocci, which are primarilystreptococci species. The bacteria excrete an extracellular slime layerthat helps anchor the bacteria to the surface and provides protectionfor the attached bacteria. Microcolony formation begins once the surfaceof the tooth has been covered with attached bacteria. The biofilm growsprimarily through cell division of adherent bacteria, rather thanthrough the attachment of new bacteria. Doubling times of bacteriaforming plaque are rapid in early development and slower in more maturebiofilms.

Coaggregation occurs when bacterial colonizers subsequently adhere tobacteria already attached to the pellicle. The result of coaggregationis the formation of a complex array of different bacteria linked to oneanother. After a few days of undisturbed plaque formation, the gingivalmargin becomes inflamed and swollen. Inflammation may result in creationof a deepened gingival sulcus. The biofilm extends into this subgingivalregion and flourishes in this protected environment, resulting in theformation of a mature subgingival plaque biofilm. Gingival inflammationdoes not appear until the biofilm changes from one composed largely ofgram-positive bacteria to one containing gram-negative anaerobes. Asubgingival bacterial microcolony, composed predominantly ofgram-negative anaerobic bacteria, becomes established in the gingivalsulcus between 3 and 12 weeks after the beginning of supragingivalplaque formation. Most bacterial species currently suspected of beingperiodontal pathogens are anaerobic, gram-negative bacteria.

Bacterial microcolonies protected within the biofilm are typicallyresistant to antibiotics (administered systemically), antiseptics ordisinfectants (administered locally), and immune defenses. Antibioticdoses that kill free-floating bacteria, for example, need to beincreased as much as 1,500 times to kill biofilm bacteria. At this highconcentration, these antimicrobials tend to be toxic to the patient aswell (see, e.g., Coghlan 1996, New Scientist 2045:32-6; Elder et al.,1995, Eye 9:102-9).

Diligent and frequent physical removal of bacterial plaque biofilms isthe most effective means of eliminating and controlling plaque. However,subgingival plaque within pockets cannot be reached by brushes, floss,or oral rinses. Therefore, frequent periodontal debridement ofsubgingival root surfaces by a dental hygienist or dentist is anessential component in prevention and treatment of periodontitis.

In certain embodiments, a microparticulate BT compound may beincorporated into oral hygiene compositions, such as but not limited to,toothpaste, mouthwash (i.e., mouth rinse), oral gels, dentifricepowders, oral sprays (including a spray dispersed by an oral inhaler),edible film, chewing gum, oral slurry, denture liquid cleaners, denturestorage liquids, and dental floss, which may be routinely used by anysubject. A microparticulate BT compound may be incorporated into oralhygiene compositions that are used primarily by dental care professions,including for example, fluoride liquid treatments, cleaningcompositions, buffing compositions, oral rinses, and dental floss. Thepresent embodiments contemplate replacement of antimicrobials formulatedwith oral hygiene compositions, which are described in the art, with thepresently described microparticulate BT compounds to provide theadvantages disclosed herein, including the range of antimicrobialactivities, solubility and bioavailability, anti-biofilm effects,non-toxicity, enhancement of antibiotic efficacies, and other propertiesas described herein.

A microparticulate BT compound may also be used for preventing ortreating caries and/or inflammation (i.e., reducing the likelihood ofoccurrence or recurrence of caries and/or inflammation, respectively) byadministering the microparticulate BT compound to the surface of theteeth. A composition comprising a microparticulate BT compound may be amucoadhesive composition that is applied to the surface of a toothand/or gum or oral mucous membrane may be in any form that adheres tosome extent to a surface or that delivers a pharmaceutically effectiveamount of the active ingredient(s) to the desired surface. Amicroparticulate BT compound can also be formulated to release slowlyfrom the composition applied to the tooth. For example, the compositionmay be a gel (e.g., a hydrogel, thiomer, aerogel, or organogel) orliquid. An organogel may comprise an organic solvent, lipoic acid,vegetable oil, or mineral oil. Such gel or liquid coating formulationsmay be applied interior or exterior to an amalgam or composite or otherrestorative composition. A slow-release composition may deliver apharmaceutically effective amount of microparticulate BT compound for 1,2, 3, 4, 5, 6, or 7 (a week) days or for 2, 3, 4, 5, 6, 7 weeks, or 1,2, 3, 4, 5, or 6 months. Such compositions can be prepared by a personskilled in the art using any number of methods known in the art.

In certain other embodiments, and as described herein, antimicrobialcompositions are provided for oral use that comprise microparticulate BTcompound and one or more additional antimicrobial compounds or agents.Particularly useful are the compositions comprising s and a secondantimicrobial agent that when administered in combination have enhancedor synergistic antimicrobial effects, as described herein. By way ofexample, an enhanced antimicrobial effect may be observed when amicroparticulate BT compound is administered together with anantimicrobial agent that chelates iron. In other particular embodiments,a microparticulate BT compound is formulated with an anti-inflammatoryagent, compound, small molecule, or macromolecule (such as a peptide orpolypeptide).

Any of the microparticulate BT compounds described herein may beformulated for oral use. In certain embodiments, microparticulate BTcompounds that are prepared with hydrophobic thiols (e.g.,thiochlorophenol) may be used and which may exhibit greater capabilitythan less hydrophobic BT compounds to adhere to teeth and tissues of themouth. BT compounds that have a net negative charge, such as thosehaving a 1:2 molar ratio (bismuth to thiol) may also have favorableadhesive properties.

The oral hygiene compositions comprising a microparticulate BT compoundmay further comprise one or more active ingredients and/or one or moreorally suitable excipients or carriers. In one embodiment, the oralhygiene compositions may further comprise baking soda or anotheralkaline compound or substance. Because of the chemical and physicalproperties of baking soda, it has wide range of applications, includingcleaning, deodorizing, and buffering. Baking soda neutralizes odorschemically, rather than masking or absorbing them. Baking soda can becombined with a microparticulate BT compound either as a mixture ofpowders, or dissolved or suspended in any one of the dentifrice powders,gels, pastes, and liquids described herein. In other embodiments, amicroparticulate BT compound can be combined with other alkali metalbicarbonate or carbonate substances (e.g., potassium bicarbonate orcalcium carbonate) that help maintain a desired alkaline pH and thatalso possess cleansing and deodorizing properties.

Oral hygiene compositions comprising a microparticulate BT compound mayfurther comprise one or more of the following ingredients. Antimicrobialagents: for example, chlorhexidine; sanguinarine extract; metronidazole;quaternary ammonium compounds (such as cetylpyridinium chloride);bis-guanides (e.g., chlorhexidine digluconate, hexetidine, octenidine,alexidine); halogenated bisphenolic compounds (e.g., 2,2′methylenebis-(4-chloro-6-bromophenol) or other phenolic antibacterialcompounds; alkylhydroxybenzoate; cationic antimicrobial peptides;aminoglycosides; quinolones; lincosamides; penicillins; cephalosporins,macrolides; tetracyclines; other antibiotics known in the art; Coleusforskohlii essential oil; silver or colloidal silver antimicrobials;tin- or copper-based antimicrobials; Manuka oil; oregano; thyme;rosemary; or other herbal extracts; and grapefruit seed extract.Anti-inflammatory or antioxidant agents: for example, ibuprofen,flurbiprofen, aspirin, indomethacin, aloe vera, turmeric, olive leafextract, cloves, panthenol, retinol, omega-3 fatty acids,gamma-linolenic acid (GLA), green tea, ginger, grape seed, etc.Anti-caries agents: for example, sodium- and stannous fluoride,aminefluorides, sodium monofluorophosphate, sodium trimetaphosphate,zinc citrate or other zinc agents, and casein. Plaque buffers: forexample, urea, calcium lactate, calcium glycerophosphate, and strontiumpolyacrylates. Vitamins: for example, Vitamins A, C and E. Plantextracts. Desensitizing agents: for example, potassium citrate,potassium chloride, potassium tartrate, potassium bicarbonate, potassiumoxalate, potassium nitrate, and strontium salts. Anti-calculus agents:for example, alkali-metal pyrophosphates, hypophosphite-containingpolymers, organic phosphonates and phosphocitrates etc. Biomolecules:for example, bacteriocins, bacteriophages, antibodies, enzymes, etc.Flavors: for example, peppermint and spearmint oils, fennel, cinnamon,etc. Proteinaceous materials: for example, collagen. Preservatives.Opacifying agents. Coloring agents. pH-adjusting agents. Sweeteningagents. Pharmaceutically acceptable carriers: for example, starch,sucrose, water or water/alcohol systems etc. Surfactants: for example,anionic, nonionic, cationic and zwitterionic or amphoteric surfactants,saponins from plant materials (see, e.g., U.S. Pat. No. 6,485,711).Particulate abrasive materials: for example, silicas, aluminas, calciumcarbonates, dicalcium phosphates, calcium pyrophosphates,hydroxyapatites, trimetaphosphates, insoluble hexametaphosphates,agglomerated particulate abrasive materials, chalk, fine ground naturalchalk and the like. Humectants: for example, glycerol, sorbitol,propyleneglycol, xylitol, lactitol etc. Binders and thickeners: forexample, sodium carboxy methyl cellulose, hydroxyethyl cellulose (soldunder the trademark NATROSOL®), xanthan gum, gum arabic, syntheticpolymers (e.g., polyacrylates and carboxyvinyl polymers such aspolyacrylate rheology modifiers sold under the trademark CARBOPOL® bythe Lubrizol Corporation). Polymeric compounds that enhance the deliveryof active ingredients such as antimicrobial agents. Buffers and salts tobuffer the pH and ionic strength of the oral care composition. Bleachingagents: for example, peroxy compounds (e.g., potassiumperoxydiphosphate). Effervescing systems: for example, sodiumbicarbonate/citric acid systems. Color change systems. In particularembodiments, an abrasive is silica or fine ground natural chalk.

The oral hygiene compositions comprising a microparticulate BT compoundthat are formulated for use as a toothpaste may further comprise ahumectant (for example, glycerol or sorbitol), a surface-active agent,binding agent, and/or a flavoring agent. The toothpastes may alsoinclude a sweetening agent, whitening agent, preservative, andantimicrobial agent. The pH of a toothpaste and other compositions fororal use is typically between pH 5.5 and 8.5. In certain embodiments,oral hygiene compositions, including toothpaste, have a pH between 7 and7.5, between 7.5 and 8, between 8 and 8.5, or between 8.5 and 9, whichmay enhance the antimicrobial activity of the microparticulate BTcompound. The toothpaste compositions described herein may include oneor more of chalk, dicalcium phosphate dihydrate, sorbitol, water,hydrated aluminum oxide, precipitated silica, sodium lauryl sulfate,sodium carboxymethyl cellulose, flavoring, sorbitan monooleate, sodiumsaccharin, tetrasodium pyrophosphate, methyl paraben, propyl paraben.One or more coloring agents, for example, FD&C Blue, can be employed ifdesired. Other suitable ingredients that may be including in atoothpaste formulation are described in the art, for example, in U.S.Pat. No. 5,560,517.

In one particular embodiment, the oral hygiene composition is amouthspray and comprises a microparticulate BT compound, an alkalinebuffer (e.g., potassium bicarbonate), an alcohol, a sweetener component,and a flavor system. The flavor system may also have or more of thefollowing: a flavorant, a humectant, a surfactant, a sweetener, and acolorant agent (see, e.g., U.S. Pat. No. 6,579,513). Surfactantsdescribed herein and known in the art for use in oral hygienecompositions may be anionic, nonionic, or amphoteric.

In another embodiment, the microparticulate BT-containing oral hygienecomposition may be combined with additional active ingredients such astaurolidine and taurultam, which have been described in the art asuseful for including in toothpastes, tooth gels, and mouthwashes fortreating treat serious infections (see, e.g., United Kingdom PatentApplication No., GB 1557163, U.S. Pat. No. 6,488,912). As describedherein, microparticulate BT can also be combined with one or moreadditional antimicrobial agents that when combined with microparticulateBT, the combination has additive or synergistic effects.

In yet another particular embodiment, an oral hygiene compositiondescribed herein may further comprise at least one or more anti-biofilmagents for controlling biofilm development, disrupting a biofilm, orreducing the amount of biofilm. As understood in the art, interspeciesquorum sensing is related to biofilm formation. Certain agents thatincrease LuxS-dependent pathway or interspecies quorum sensing signal(see, e.g., U.S. Pat. No. 7,427,408) contribute to controllingdevelopment and/or proliferation of a biofilm. Exemplary agents include,by way of example, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL)blocking compounds and N-butyryl-L-homoserine lactone (BHL) analogs,either in combination or separately (see, e.g., U.S. Pat. No.6,455,031). An oral hygiene composition comprising a microparticulate BTcompound and at least one anti-biofilm agent can be delivered locallyfor disruption and inhibition of bacterial biofilm and for treatment ofperiodontal disease (see, e.g., U.S. Pat. No. 6,726,898).

An oral hygiene composition described herein may contain a sufficientamount of a microparticulate BT compound that effects substantialantimicrobial action during the time required for a normal toothbrushing, mouth rinsing, or flossing. As described herein amicroparticulate BT compound may be retained on oral surfaces (such astooth, amalgam, composite, mucous membrane, gums). A microparticulate BTcompound retained on the teeth and gums after completion of brushing,rinsing, flossing, for example, may continue to provide extendedanti-biofilm and anti-inflammatory action.

In other embodiments, microparticulate BT compounds are slowly releasedfrom muco-adhesive polymers or other agents that contribute to retentionof microparticulate BT compound on mucosal and tooth surfaces.Microparticulate BT compounds may be added to stable, viscous,mucoadhesive aqueous compositions, which may also be used for theprevention and treatment of ulcerative, inflammatory, and/or erosivedisorders of mucous membranes and/or the delivery of pharmaceuticallyactive compounds to mucosal surfaces for topical treatment or transferto the systemic circulation (see, e.g., U.S. Pat. No. 7,547,433).

In another embodiment, oral hygiene compositions comprising amicroparticulate BT compound further comprise olive oil, which mayenhance plaque removal. The use of olive oil in a product intended fororal hygiene, such as a toothpaste, a mouthwash, a spray, oral inhaler,or chewing gum, may contribute to elimination or reduction (a decrease)of bacterial plaque and/or to elimination or reduction (decrease of) inthe numbers of bacteria present in the buccal cavity, thereby achievinga reduction in the occurrence of dental diseases (e.g., tooth decay,periodontal disease) and halitosis (see, e.g., U.S. Pat. No. 7,074,391).

In other embodiments, an oral hygiene composition comprising amicroparticulate BT compound may further comprise a mucosal disinfectantpreparation for topical application in the mouth. An oral hygienecomposition may further comprise an aqueous slurry useful for cleaningthe tongue and throat (see, e.g., U.S. Pat. No. 6,861,049). In stillanother embodiment, an oral hygiene composition comprising amicroparticulate BT compound may further comprise at least one mint thatis used for preventing (i.e., reducing the likelihood of occurrence)formation of a cavity (dental caries) or reducing the number ofcavities. One such mint, containing an arginine bicarbonate calciumcarbonate complex and sold under the trademark CAVISTAT® (OrtekTherapeutics, Inc., Roslyn Heights, N.Y.), contains arginine andcalcium, which helps neutralize acid pH and promotes adherence ofcalcium to enamel surfaces. The inclusion of mint in an oral hygienecomposition comprising a microparticulate BT compound may thus increasepH and enhance adherence of a microparticulate BT compound to oralsurfaces.

Compositions Comprising Microparticulate Bismuth-Thiols Formulated forOrthopedic Use.

In a particular embodiment, methods are provided for using compositionscomprising a microparticulate BT compound for preventing and/or treatingmicrobial infections and inflammation resulting from an orthopedicprocedure (e.g., orthopedic surgery, orthopedic therapy, arthroplasty(including two-step arthroplasty), orthodontic therapy). Thecompositions comprising microparticulate BT compounds as describedherein are therefore useful for preventing and/or treating (i.e.,reducing or inhibiting development of, reducing the likelihood ofoccurrence or recurrence of) microbial infections of the skeleton andsupporting structure (i.e., bones, joints, muscles, ligaments, tendons)such as osteomyelitis. The compositions described herein comprising amicroparticulate BT compound may also be useful for preventing and/orcontrolling (i.e., slowing, retarding, inhibiting) biofilm development,disrupting a biofilm, or reducing the amount of biofilm present in ajoint or on the surface of a bone, ligament, tendon, or tooth.

The compositions described herein for orthopedic use that comprise amicroparticulate BT compound may further comprise one or more additionalantimicrobial compounds or agents. Particularly useful are thecompositions comprising a microparticulate BT compound and a secondantimicrobial agent that when administered in combination have enhancedor synergistic antimicrobial effects, as described herein. By way of anadditional example, an enhanced antimicrobial effect may be observedwhen a microparticulate BT compound is administered together with anantimicrobial agent that chelates iron. In other particular embodiments,a microparticulate BT compound is formulated with an anti-inflammatoryagent, compound, small molecule, or macromolecule (such as a peptide orpolypeptide).

Compositions comprising a microparticulate BT compound may be combinedwith at least one other antimicrobial agent (i.e., a second, third,fourth, etc. antimicrobial agent) that when administered in combinationhave enhanced or synergistic antimicrobial effects (i.e., greater thanan additive effect). By way of example, an enhanced antimicrobial effectmay be observed when a microparticulate BT compound is administeredtogether with an antimicrobial agent that chelates iron. In particularembodiments, compositions comprising a microparticulate BT compound maybe combined with at least one other antimicrobial agent and/oranti-inflammatory agent selected from the following: Antimicrobialagents: for example, chlorhexidine; sanguinarine extract; metronidazole;quaternary ammonium compounds (such as cetylpyridinium chloride);bis-guanides (e.g., chlorhexidine digluconate, hexetidine, octenidine,alexidine); halogenated bisphenolic compounds (e.g., 2,2′methylenebis-(4-chloro-6-bromophenol) or other phenolic antibacterialcompounds; alkylhydroxybenzoate; cationic antimicrobial peptides;aminoglycosides; quinolones; lincosamides; penicillins; cephalosporins,macrolides; tetracyclines; other antibiotics known in the art; Coleusforskohlii essential oil; silver or colloidal silver antimicrobials;tin- or copper-based antimicrobials; Manuka oil; oregano; thyme;rosemary; or other herbal extracts; and grapefruit seed extract.Anti-inflammatory or antioxidant agents: for example, ibuprofen,flurbiprofen, aspirin, indomethacin, aloe vera, turmeric, olive leafextract, cloves, panthenol, retinol, omega-3 fatty acids,gamma-linolenic acid (GLA), green tea, ginger, grape seed, etc. Inparticular embodiments, the compositions comprising microparticulate BTcompound may further comprise an antibiotic selected from clindamycin,vancomycin, daptomycin, cefazolin, gentamicin, tobramycin,metronidazole, cefaclor, ciprofloxacin, or other antimicrobial such as aquaternary ammonium compound (e.g., benzalkonium chloride, cetylpyridinium chloride), an anti-microbial zeolite, alkali metal hydroxide,or an alkaline earth metal oxide. The compositions may optionallycomprise one or more pharmaceutically suitable carriers (i.e.,excipients), surfactants, buffers, diluents, and salts, and bleachingagents, which are described herein. Accordingly, these and certain ofthe related herein disclosed embodiments contemplate inclusion in suchproducts and processes of the presently disclosed microparticulate BTcompositions, which may include one or more microparticulate BT, andwhich may also optionally further include an antibiotic such as asynergizing or an enhancing antibiotic as described herein.

Biological, Biomedical and Other Uses for Microparticulate BTs.

Certain other embodiments contemplate use of the herein describedmicroparticulate BTs, whether as individual BTs or BTs in which thebismuth moiety is replaced with a different Group V metal such asantimony (Sb) or arsenic (As), and/or as such BTs in combination withone or more antibiotic with which, as described herein, the BT exhibitssynergizing or enhanced antimicrobial activity, in orally ingestednutritional formulations.

According to non-limiting theory, the inclusion of microparticulate BTsin such formulations along with other components such as vitamins,minerals, amino acids, hydrocarbons including carbohydrates, fattyacids, oils, phytonutrients, teas, herbs or herbal extracts, and/orother nutritional or food products, may in certain embodiments result inthe blockage or retardation of nutrient uptake by microbial populationsin the gastrointestinal tract, in a manner that promotes increased(e.g., in a statistically significant manner relative to an appropriatecontrol) bioavailability of the BT and optionally the antibiotic and/orof the additional nutritional component(s) to the host digestive tract.In certain other embodiments, by varying the particular vitamins,minerals, amino acids, hydrocarbons including carbohydrates, fattyacids, oils, phytonutrients, teas, herbs or herbal extracts, and/orother nutritional or food products that are included in the oralmicroparticulate BT (or AsT or SbT) formulation, bioavailability of theBT and optionally of the antibiotic and/or of the additional nutritionalcomponent(s) to the host digestive tract may be decreased (e.g., in astatistically significant manner relative to an appropriate control).

For instance, it may be desirable when a pathologic gastrointestinal(GI) tract infection is present to administer a microparticulate BTformulation that discourages intestinal absorption of the BT compoundsso that they remain bioavailable within the GI tract in order to exertantimicrobial effects against the infectious pathogens. Those familiarwith the art will be aware of a number of vitamins, minerals, aminoacids, hydrocarbons including carbohydrates, fatty acids, oils,phytonutrients, teas, herbs and/or herbal extracts that promote ordiscourage GI tract absorption of nutrients, such that formulations forincreasing or decreasing the GI tract presence of one or more components(e.g., the microparticulate BT compound, the antibiotic, or one or moreparticular nutrients) may be prepared using the presently disclosedmicroparticulate BTs (or AsT or SbT).

Certain other embodiments provided herein contemplate inclusion of thepresently disclosed microparticulate BT compounds in compositions fororal delivery to reduce fecal or digestive gas odors, for instance inpatients who have undergone colostomy, and in other compositions fortopical delivery to reduce underarm, foot or other body odors associatedwith topical microbial presence. A number of skin and GI tract microbialpopulations, including planktonic and biofilm bacteria, are susceptibleto low concentrations of the herein described microparticulate BTcompounds, including such BT compounds when present with enhancing orsynergizing antibiotics as described herein.

Accordingly, certain embodiments contemplate orally delivered andtopically delivered microparticulate BT formulations to decrease (e.g.,in a statistically significant manner relative to an appropriatecontrol) populations of GI-resident or skin-resident bacteria in amanner that reduces or alleviates the problem of unwanted odor. Oral andtopical pharmaceutical formulations are described below, such that theseand related embodiments offer advantages associated with the presentmicroparticulate formulation of BT, such as compatible bioavailabilityand solubility properties and low toxicity; other factors that mayinfluence the selection of antimicrobial compositions are describedelsewhere herein and may also be found, e.g., in U.S. Pat. No.6,582,719.

An exemplary BT compound, BisEDT, has been applied (50 uL of a 1 mg/mLsolution in DMSO) to the axillary area in human test subjects and shownto neutralize body odors for two to three days. A mixture of BisEDT intalcum powder applied to the feet of a human test subject substantiallyreduced foot odor. Laboratory mice fed 1 mg/kg BisEDT orally twice dailyfor five days exhibited 90% reductions in the number of fecal flora.Related embodiments also contemplate a generally useful deodorant forany thiol-containing solution that emits odors (e.g., fish oils such assalmon oil), comprising a microparticulate BT preparation as describedherein that is made with an excess of bismuth, and that can be added tothe thiol-containing solution as an odor quenching agent. The resultingmixture retains the antimicrobial properties of the microparticulate BT.Other contemplated applications include solvents such as otherbiological source oils or butters, for instance, hemp oil, tea tree oil,shea butter, flax seed oil, fish oils, and in certain embodiments suchoils as may have an independent or synergistic anti-inflammatory and/orpain-reducing and/or other beneficial physiologic effect.

Pharmaceutical Compositions and Administration

Certain embodiments also relate to a pharmaceutical compositioncontaining the microparticulate BT compounds disclosed herein; incertain such embodiments the pharmaceutical composition may furthercomprise one or more antibiotics such as an antibiotic with which the BTcompound exhibits a synergizing or enhancing effect as described herein.In one embodiment, there is provided a composition comprising one ormore such microparticulate BT compounds in a pharmaceutically acceptablecarrier, excipient or diluent and in a therapeutic amount, as disclosedherein, when administered to an animal, preferably a mammal, mostpreferably a human patient.

Administration of the microparticulate BT compounds, or theirpharmaceutically acceptable salts, in pure form or in an appropriatepharmaceutical composition, can be carried out via any of the acceptedmodes of administration of agents for serving similar utilities. Thepharmaceutical compositions can be prepared by combining amicroparticulate BT compound with an appropriate pharmaceuticallyacceptable carrier, diluent or excipient, and may be formulated intopreparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.Typical routes of administering such pharmaceutical compositionsinclude, without limitation, oral, topical, transdermal, inhalation,parenteral, sublingual, rectal, vaginal, and intranasal. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques.Pharmaceutical compositions are formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa subject or patient take the form of one or more dosage units, wherefor example, a tablet may be a single dosage unit, and a container of acompound in aerosol form may hold a plurality of dosage units. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this art; for example, see The Science and Practiceof Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science,2000). The composition to be administered will, in any event, contain atherapeutically effective amount of a compound, or a pharmaceuticallyacceptable salt thereof, for treatment of a disease or condition ofinterest in accordance with the teachings herein.

The pharmaceutical compositions useful herein also contain apharmaceutically acceptable carrier, including any suitable diluent orexcipient, which includes any pharmaceutical agent that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition, and which may be administered without undue toxicity.Pharmaceutically acceptable carriers include, but are not limited to,liquids, such as water, saline, glycerol and ethanol, and the like. Athorough discussion of pharmaceutically acceptable carriers, diluents,and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES(Mack Pub. Co., N.J. current edition).

A pharmaceutical composition may be in the form of a solid or liquid. Inone aspect, the carrier(s) are particulate, so that the compositionsare, for example, in tablet or powder form. The carrier(s) may beliquid, with the compositions being, for example, an oral syrup,injectable liquid or an aerosol, which is useful in, for example,inhalatory administration.

When intended for oral administration, the pharmaceutical composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, sodium starch glycolate sold under the trademarkPRIMOGEL™ (DMV International), corn starch and the like; lubricants suchas magnesium stearate or vegetable stearine sold under the trademarkSTEROTEX® (Abitec Corp.); glidants such as colloidal silicon dioxide;sweetening agents such as honey, sucrose or saccharin; a flavoring agentsuch as peppermint, methyl salicylate or orange flavoring; and acoloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions, whether they be solutions,suspensions or other like form, may include one or more of the followingadjuvants: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or diglycerides whichmay serve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Physiological saline isa preferred adjuvant. An injectable pharmaceutical composition ispreferably sterile.

A liquid pharmaceutical composition intended for either parenteral ororal administration should contain an amount of a microparticulate BTcompound such that a suitable dosage will be obtained. Typically, thisamount is at least 0.01% of a microparticulate BT compound in thecomposition. When intended for oral administration, this amount may bevaried to be between 0.1 and about 70% of the weight of the composition.Preferred oral pharmaceutical compositions contain between about 4% andabout 50% of the BT compound. Preferred pharmaceutical compositions andpreparations according to the present invention are prepared so that aparenteral dosage unit contains between 0.01 to 10% by weight of themicroparticulate BT compound prior to dilution.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, shea butter, tea tree oil, flax seed oil,hemp oil or other plant or vegetable oils including those known to haveanti-inflammatory and/or anti-pain or other beneficial effects, salmonoil or other fish oils including those known to have anti-inflammatoryand/or anti-pain or other beneficial effects, diluents such as water andalcohol, and emulsifiers and stabilizers. Thickening agents may bepresent in a pharmaceutical composition for topical administration. Ifintended for transdermal administration, the composition may include atransdermal patch or iontophoresis device. Topical formulations maycontain a concentration of the microparticulate BT compound from about0.1 to about 10% w/v (weight per unit volume).

The pharmaceutical composition may be intended for rectaladministration, in the form, for example, of a suppository, which willmelt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition may include various materials, whichmodify the physical form of a solid or liquid dosage unit. For example,the composition may include materials that form a coating shell aroundthe active ingredients. The materials that form the coating shell aretypically inert, and may be selected from, for example, sugar, shellac,and other enteric coating agents. Alternatively, the active ingredientsmay be encased in a gelatin capsule.

The pharmaceutical composition in solid or liquid form may include anagent that binds to the microparticulate BT compound and thereby assistsin the delivery of the compound. Suitable agents that may act in thiscapacity include a monoclonal or polyclonal antibody, a protein or aliposome. Certain contemplated embodiments, however, expressly excludethe inclusion of a liposome in the pharmaceutical composition.

The pharmaceutical composition may consist of dosage units that can beadministered as an aerosol. The term aerosol is used to denote a varietyof systems ranging from those of colloidal nature to systems consistingof pressurized packages. Delivery may be by a liquefied or compressedgas or by a suitable pump system that dispenses the active ingredients.Aerosols of the microparticulate BT compounds may be delivered in singlephase, bi-phasic, or tri-phasic systems in order to deliver the activeingredient(s). Delivery of the aerosol includes the necessary container,activators, valves, subcontainers, and the like, which together may forma kit. One skilled in the art, without undue experimentation maydetermine preferred aerosols.

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art. For example, a pharmaceuticalcomposition intended to be administered by injection can be prepared bycombining a compound of the invention with sterile, distilled water soas to form a solution. A surfactant may be added to facilitate theformation of a homogeneous solution or suspension. Surfactants arecompounds that non-covalently interact with the compound of theinvention so as to facilitate dissolution or homogeneous suspension ofthe compound in the aqueous delivery system.

The herein described microparticulate BT compounds, or theirpharmaceutically acceptable salts, are administered in a therapeuticallyeffective amount, which will vary depending upon a variety of factorsincluding the activity of the specific compound employed; the metabolicstability and length of action of the compound; the age, body weight,general health, sex, and diet of the patient; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy. Generally, a therapeutically effective daily dose is(for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (fora 70 kg mammal) from about 0.01 mg/kg (i.e., 7 mg) to about 50 mg/kg(i.e., 3.5 g); more preferably a therapeutically effective dose is (fora 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg(i.e., 1.75 g).

The ranges of effective doses provided herein are not intended to belimiting and represent preferred dose ranges. However, the mostpreferred dosage will be tailored to the individual subject, as isunderstood and determinable by one skilled in the relevant arts. (see,e.g., Berkow et al., eds., The Merck Manual, 16^(th) edition, Merck andCo., Rahway, N.J., 1992; Goodmanetna., eds., Goodman and Cilman's ThePharmacological Basis of Therapeutics, 10^(th) edition, Pergamon Press,Inc., Elmsford, N.Y., (2001); Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi,Pharmacology, Little, Brown and Co., Boston, (1985); Osolci al., eds.,Remington's Pharmaceutical Sciences, 18^(th) edition, Mack PublishingCo., Easton, Pa. (1990); Katzung, Basic and Clinical Pharmacology,Appleton and Lange, Norwalk, Conn. (1992)).

The total dose required for each treatment can be administered bymultiple doses or in a single dose over the course of the day, ifdesired. Generally, treatment is initiated with smaller dosages, whichare less than the optimum dose of the compound. Thereafter, the dosageis increased by small increments until the optimum effect under thecircumstances is reached. The diagnostic pharmaceutical compound orcomposition can be administered alone or in conjunction with otherdiagnostics and/or pharmaceuticals directed to the pathology, ordirected to other symptoms of the pathology. The recipients ofadministration of microparticulate BT compounds and/or compositions canbe any vertebrate animal, such as mammals. Among mammals, the preferredrecipients are mammals of the Orders Primate (including humans, apes andmonkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs),Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora(including cats, and dogs). Among birds, the preferred recipients areturkeys, chickens and other members of the same order. The mostpreferred recipients are humans.

For topical applications, it is preferred to administer an effectiveamount of a microparticulate BT-containing pharmaceutical composition toa target area, e.g., skin surfaces, mucous membranes, and the like. Thisamount will generally range from about 0.0001 mg to about 1 g of a BTcompound per application, depending upon the area to be treated, whetherthe use is diagnostic, prophylactic or therapeutic, the severity of thesymptoms, and the nature of the topical vehicle employed. A preferredtopical preparation is an ointment, wherein about 0.001 to about 50 mgof active ingredient is used per cc of ointment base. The pharmaceuticalcomposition can be formulated as transdermal compositions or transdermaldelivery devices (“patches”). Such compositions include, for example, abacking, active compound reservoir, a control membrane, liner andcontact adhesive. Such transdermal patches may be used to providecontinuous pulsatile, or on demand delivery of the compounds of thepresent invention as desired.

The microparticulate BT compositions can be formulated so as to providequick, sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.Controlled release drug delivery systems include osmotic pump systemsand dissolutional systems containing polymer-coated reservoirs ordrug-polymer matrix formulations. Examples of controlled release systemsare given in U.S. Pat. Nos. 3,845,770 and 4,326,525 and in P. J. Kuzmaet al, Regional Anesthesia 22 (6): 543-551 (1997), all of which areincorporated herein by reference.

The microparticulate BT compositions can also be delivered throughintranasal drug delivery systems for local, systemic, and nose-to-brainmedical therapies. Nasal, respiratory and sinus drug delivery technologysold under the trademark CONTROLLED PARTICLE DISPERSION® (KurveTechnology, Inc.) (CPD)™, traditional nasal spray bottles, inhalers ornebulizers are known by those skilled in the art to provide effectivelocal and systemic delivery of drugs by targeting the olfactory regionand paranasal sinuses.

The invention also relates in certain embodiments to an intravaginalshell or core drug delivery device suitable for administration to thehuman or animal female. The device may be comprised of the activepharmaceutical ingredient in a polymer matrix, surrounded by a sheath,and capable of releasing the compound in a substantially zero orderpattern on a daily basis similar to devices used to apply testosteroneas described in WO 98/50016.

Current methods for ocular delivery include topical administration (eyedrops), subconjunctival injections, periocular injections, intravitrealinjections, surgical implants and iontophoresis (uses a small electricalcurrent to transportionized drugs into and through body tissues). Thoseskilled in the art would combine the best suited excipients with thecompound for safe and effective intra-occular administration.

The most suitable route will depend on the nature and severity of thecondition being treated. Those skilled in the art are also familiar withdetermining administration methods (oral, intravenous, inhalation,sub-cutaneous, rectal etc.), dosage forms, suitable pharmaceuticalexcipients and other matters relevant to the delivery of the compoundsto a subject in need thereof.

The presently described compositions and methods may also find use inthe treatment of acute and chronic wounds and wound biofilms, including,for example, as burn creams, as topicals for the treatment of existingwounds including those described herein, for prevention of chronicwounds, for treatment of MRSA skin infections, and for other relatedindications as disclosed herein and as will be apparent to the skilledperson in view of the present disclosure.

Non-limiting examples of bacteria against which the herein describedcompositions and methods may find beneficial use, according to certainembodiments as described herein, include Staphylococcus aureus (S.aureus), MRSA (methicillin-resistant S. aureus), Staphylococcusepidermidis, MRSE (methicillin-resistant S. epidermidis), Mycobacteriumtuberculosis, Mycobacterium avium, Pseudomonas aeruginosa,drug-resistant P. aeruginosa, Escherichia coli, enterotoxigenic E. coli,enterohemorrhagic E. coli, Klebsiella pneumoniae, Clostridium difficile,Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis,methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae,Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica,Vibrio cholera, Shigella flexneri, vancomycin-resistant Enterococcus(VRE), Burkholderia cepacia complex, Francisella tularensis, Bacillusanthracis, Yersinia pestis, Pseudomonas aeruginosa, vancomycin-sensitiveand vancomycin-resistant enterococci (e.g., E. faecalis, E. faecium),methicillin-sensitive and methicillin-resistant staphylococci (e.g., S.aureus, S. epidermidis) and Acinetobacter baumannii, Staphylococcushaemolyticus, Staphylococcus hominis, Enterococcus faecium,Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis,Klebsiella pneumonia, Proteus mirabilis, Proteus vulgaris, Yersiniaenterocolytica, Stenotrophomonas maltophilia, Streptococcus pneumonia,penicillin-resistant Streptococcus pneumonia, Burkholderia cepacia,Bukholderia multivorans, Mycobacterium smegmatis and E. cloacae.

The practice of certain embodiments of the present invention willemploy, unless indicated specifically to the contrary, conventionalmethods of microbiology, molecular biology, biochemistry, cell biology,virology and immunology techniques that are within the skill of the art,and reference to several of which is made below for the purpose ofillustration. Such techniques are explained fully in the literature.See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (2ndEdition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual(1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984).

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” or “an aspect” means that a particular feature, structure orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present invention. Thus, theappearances of the phrases “in one embodiment” or “in an embodiment” invarious places throughout this specification are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more embodiments.

Certain embodiments relate to methods, compositions and kits fortreating an acute or chronic wound or a wound biofilm in a subject,which may comprise promoting skin tissue repair in the subject, or foraltering one or more cellular wound repair activity in a cell orplurality of cells. A cell generally indicates a single cell, whereas aplurality of cells indicates more than one cell. The cells may comprisea tissue, organ or entire organism. Furthermore, the cell or cells maybe located in vivo, in vitro, or ex vivo. Maintaining cell, tissue andorgan cultures are routine procedures for one of skill in the art, theconditions and media for which can be easily ascertained. (See, forexample, Freshney, Culture of Animal Cells: A Manual of Basic Technique,Wiley-Liss 5^(th) Ed. (2005); Davis, Basic Cell Culture, OxfordUniversity Press 2^(nd) Ed. (2002)).

As disclosed herein, certain embodiments relate to methods for treatingan acute or chronic wound or a wound biofilm in a subject that comprisesadministering to the subject a therapeutically effective amount of acomposition comprising a BT compound as described herein for use in suchmethod (e.g., as provided in the form of a plurality of substantiallymonodisperse microparticles), and optionally in certain furtherembodiments also comprising an antibiotic compound as described hereinfor use in such method, for example, a BT compound such as BisEDT orBisBAL or other compounds presented in Table 1 herein, or any other BTagent such as those described in Domenico et al. (1997 Antimicrob.Agent. Chemother. 41:1697; 2001 Antimicrob. Agent. Chemother. 45:1421)and/or in U.S. RE37,793, U.S. Pat. No. 6,248,371, U.S. Pat. No.6,086,921, and U.S. Pat. No. 6,380,248 and/or as prepared according tothe methods disclosed herein. Certain other embodiments relate tomethods that comprise contacting any natural surface with a compositioncomprising one or more of the herein described microparticulate BTcompounds, where such step of contacting may comprise one or more ofdirectly applying, coating, dipping, irrigating, spraying, painting orotherwise bringing the BT composition into contact with the naturalsurface.

The step of administering to a subject such as a human or othermammalian subject may be performed by any means known to the art, forexample, topically (including via direct administration to skin or toany epithelial tissue surface, including such surfaces as may be presentin glandular tissues or in the respiratory and/or gastrointestinaltracts), vaginally, intraperitoneally, orally, parenterally,intravenously, intraarterially, transdermally, sublingually,subcutaneously, intramuscularly, transbuccally, intranasally, viainhalation, intraoccularly, subcutaneously, intraadiposally,intraarticularly or intrathecally.

In preferred embodiments administering may be performed topically, wherepharmaceutical excipients or carriers for topical use are describedherein and known in the art.

As noted above, certain invention embodiments described herein relate totopical formulations of the described BT compounds (e.g., BisEDT and/orBisBAL), which formulations may in certain further embodiments compriseone or more antibiotic compounds as described herein, for instance,amikacin, ampicillin, cefazolin, cefepime, chloramphenicol,ciprofloxacin, clindamycin (or another lincosamide antibiotic),daptomycin (sold under the trademark CUBICIN®), doxycycline,gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid (sold underthe trademark ZYVOX®), minocycline, nafcilin, paromomycin, rifampin,sulphamethoxazole, tobramycin and vancomycin; or a carbapenemantibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, aglycopeptide antibiotic, a lincosamide antibiotic, apenicillinase-resistant penicillin antibiotic, and/or an aminopenicillinantibiotic, and/or an aminoglycoside antibiotic such as amikacin,arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin or apramycin, and/or alipopeptide antibiotic such as daptomycin (CUBICIN®), or anoxazolidinone antibiotic such as linezolid (ZYVOX®). These and relatedformulations may comprise the BT compound(s) (and optionally one or moreantibiotics) in a pharmaceutically acceptable carrier, excipient ordiluent and in a therapeutic amount, as disclosed herein, whenadministered topically to an animal, preferably a mammal, and mostpreferably a human, and in particularly preferred embodiments, a humanhaving an acute or chronic wound or a wound that contains a bacterialinfection which may be biofilm-related (e.g., in which bacteria capableof promoting biofilm formation may be present but a biofilm is not yetdetectable) or that contains a bacterial infection such as a biofilm orother bacterial presence.

Topical administration of the BT compounds described herein, or theirpharmaceutically acceptable salts, in pure form or in an appropriatepharmaceutical composition, can be carried out via any of the acceptedmodes of topical administration of agents for serving similar utilities.Topical application or administration of a composition includes, inpreferred embodiments, directly contacting the composition (e.g., atopical formulation) with skin and/or another epithelial tissue surface(e.g., respiratory tract, gastrointestinal tract and/or glandularepithelial linings) of the subject undergoing treatment, which may be atone or more localized or widely distributed skin and/or other epithelialtissue surface sites and which may generally refer to contacting thetopical formulation with an acute or chronic wound site that issurrounded by intact stratum corneum or epidermis but need not be solimited; for instance, certain embodiments contemplate as a topicalapplication the administration of a topical formulation described hereinto injured, abraded or damaged skin, or skin of a subject undergoingsurgery, such that contact of the topical formulation may take place notonly with stratum corneum or epidermis but also with skin granular cell,spinous cell, and/or basal cell layers, and/or with dermal or underlyingtissues, for example, as may accompany certain types of wound repair orwound healing or other skin tissue remodeling.

Such skin tissue repair may therefore comprise, in certain preferredembodiments, dermal wound healing, as may be desirable, for example, inpreventing or ameliorating an acute chronic wound or a wound biofilm or,as another example, in preventing or ameliorating skin wound dehiscence,or in improving, accelerating or otherwise enhancing dermal woundhealing when an acute or chronic wound and/or skin wound dehiscence maybe present. Certain other embodiments that contemplate topicaladministration to an epithelial tissue surface present in respiratorytract, gastrointestinal tract and/or glandular linings similarly maycomprise administration of the topical formulation by an appropriateroute as will be known in the art for delivering a topical preparationas provided herein, to one or more epithelial tissue surfaces present inrespiratory (e.g., airway, nasopharyngeal and laryngeal paths, tracheal,pulmonary, bronchi, bronchioles, alveoli, etc.) and/or gastrointestinal(e.g., buccal, esophageal, gastric, intestinal, rectal, anal, etc.)tracts, and/or other epithelial surfaces.

According to certain contemplated embodiments topical administration maycomprise direct application into an open wound. For instance, an openfracture or other open wound may include a break in the skin that mayexpose additional underlying tissues to the external environment in amanner that renders them susceptible to microbial infection. Such asituation is not uncommon in certain types of acute traumatic militarywounds, including, for example, Type III (severe) open fractures. Inaccord with these and related embodiments, topical administration may beby direct contact of the herein described BT composition with suchdamaged skin and/or another epithelial surface and/or with othertissues, such as, for instance, connective tissues including muscle,ligaments, tendons, bones, circulatory tissues such as blood vessels,associated nerve tissues, and any other organs that may be exposed insuch open wounds. Examples of other tissues that may be exposed, andhence for which such direct contact is contemplated, include kidney,bladder, liver, pancreas, and any other tissue or organ that may be sodetrimentally exposed to opportunistic infection in relation to an openwound.

The topical formulations (e.g., pharmaceutical compositions) may beprepared by combining the described BT compound (e.g., comprising acompound described in U.S. RE37,793, U.S. Pat. No. 6,248,371, U.S. Pat.No. 6,086,921, and/or U.S. Pat. No. 6,380,248 and/or prepared accordingto the present disclosure such as the herein described microparticulateBT suspensions), and in certain related embodiments by combining one ormore desired antibiotics (e.g., an aminoglycoside antibiotic such asamikacin) separately or together with the BT compound, with anappropriate pharmaceutically acceptable carrier, diluent or excipientfor use in a topical formulation preparation, and may be formulated intopreparations in solid, semi-solid, gel, cream, colloid, suspension orliquid or other topically applied forms, such as powders, granules,ointments, solutions, washes, gels, pastes, plasters, paints,bioadhesives, microsphere suspensions, and aerosol sprays.

Pharmaceutical compositions of these and related embodiments areformulated so as to allow the active ingredients contained therein, andin particularly preferred embodiments the herein described BTcompound(s) alone or in combination with one or more desired antibiotics(e.g., a carbapenem antibiotic, a cephalosporin antibiotic, afluoroquinolone antibiotic, a glycopeptide antibiotic, a lincosamideantibiotic, a penicillinase-resistant penicillin antibiotic, and anaminopenicillin antibiotic, or an aminoglycoside antibiotic such asamikacin, or rifamycin) which may be applied simultaneously orsequentially and in either order, to be bioavailable upon topicaladministration of the formulation containing the BT compound(s) and/orantibiotic composition(s) to an acute or chronic wound and optionally tosurrounding skin of a subject, such as a mammal, including a human, andin certain preferred embodiments a human patient having an acute orchronic wound, or being at increased risk for having, an acute orchronic wound or a wound biofilm or wound dehiscence (e.g., an obeseand/or diabetic individual). Certain embodiments disclosed hereincontemplate topical administration of a BT compound and of anantibiotic, including administration that may be simultaneous orsequential and in either order, but the invention is not intended to beso limited and in other embodiments expressly contemplates a distinctroute of administration for the BT compound relative to the route ofadministration of the antibiotic. Thus, the antibiotic may beadministered orally, intravenously, or by any other route ofadministration as described herein, while the BT compound may beadministered by a route that is independent of the route used for theantibiotic. As a non-limiting, illustrative example, the BT compound maybe administered topically as provided herein, while the antibiotic maybe simultaneously or sequentially (and in any order) administered by adistinct route, such as orally, intravenously, transdermally,subcutaneously, intramuscularly and/or by any other route ofadministration.

The topical formulations described herein deliver a therapeuticallyeffective amount of the antiseptic or wound-healing agent(s) (andoptionally the antibiotic(s)) to the wound site, for instance, to skincells such as dermal fibroblasts. Preferred formulations may becontacted with a desired site such as a topical wound site, a chronicwound, an epithelial tissue surface or other intended site ofadministration by spraying, irrigating, dipping and/or painting; suchformulations therefore may exhibit ready permeability into the skin, ascan be determined according to any of a number of establishedmethodologies known to the art for testing the skin permeability of adrug composition (see, e.g., Wagner et al., 2002 J. Invest. Dermatol.118:540, and references cited therein; Bronaugh et al., 1985 J. Pharm.Sci. 74:64; Bosman et al., 1998 J. Pharm. Biomed. Anal. 17:493-499;Bosman et al., 1996 J. Pharm Biomed Anal. 1996 14:1015-23; Bonferoni etal., 1999 Pharm. Dev. Technol. 4:45-53; Frantz, Instrumentation andmethodology for in vitro skin diffusion cells in methodology for skinabsorption. In: Methods for Skin Absorption (Kemppainen & Reifenrath,Eds), CRC Press, Florida, 1990, pp. 35-59; Tojo, Design and calibrationof in vitro permeation apparatus. In: Transdermal Controlled SystemicMedications (Chien Y W, Ed), Marcel Dekker, New York, 1987, 127-158;Barry, Methods for studying percutaneous absorption. In: DermatologicalFormulations: Percutaneous absorption, Marcel Dekker, New York, 1983,234-295).

Compositions, and formulations comprising such compositions, that willbe administered to the skin of a subject or patient may in certainembodiments take the form of one or more dosage units, where forexample, a liquid-filled capsule or ampule may contain a single dosageunit, and a container of a topical formulation as described herein inaerosol form may hold a plurality of dosage units. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see The Science and Practice ofPharmacy, 20th Edition (Philadelphia College of Pharmacy and Science,2000). The composition or formulation to be administered will, in anyevent, contain a therapeutically effective amount of an antisepticand/or wound healing-promoting compound as provided herein (e.g., a BTcompound), or a pharmaceutically acceptable salt thereof, in accordancewith the present teachings.

As noted above, the present topical formulations may take any of a widevariety of forms, and include, for example, creams, lotions, solutions,sprays, gels, ointments, pastes or the like, and/or may be prepared soas to contain liposomes, micelles, and/or microspheres. See, e.g., U.S.Pat. No. 7,205,003. For instance, creams, as is well known in the artsof pharmaceutical and cosmeceutical formulation, are viscous liquids orsemisolid emulsions, either oil-in-water or water-in-oil. Cream basesare water-washable, and contain an oil phase, an emulsifier, and anaqueous phase. The oil phase, also called the “internal” phase, isgenerally comprised of petrolatum and a fatty alcohol such as cetyl orstearyl alcohol. The aqueous phase usually, although not necessarily,exceeds the oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation is generally a nonionic, anionic,cationic or amphoteric surfactant.

Lotions, which are preferred for delivery of cosmetic agents, arepreparations to be applied to the skin surface without friction, and aretypically liquid or semi-liquid preparations in which solid particles,including the active agent, are present in a water or alcohol base.Lotions are usually suspensions of solids, and preferably comprise aliquid oily emulsion of the oil-in-water type. Lotions are preferredformulations herein for treating large body areas, because of the easeof applying a more fluid composition. It is generally preferred that theinsoluble matter in a lotion be finely divided. Lotions will typicallycontain suspending agents to produce better dispersions as well ascompounds useful for localizing and holding the active agent in contactwith the skin, e.g., methylcellulose, sodium carboxymethyl-cellulose, orthe like.

Solutions are homogeneous mixtures prepared by dissolving one or morechemical substances (solutes) in a liquid such that the molecules of thedissolved substance are dispersed among those of the solvent. Thesolution may contain other pharmaceutically acceptable and/orcosmeceutically acceptable chemicals to buffer, stabilize or preservethe solute. Common examples of solvents used in preparing solutions areethanol, water, propylene glycol or any other pharmaceuticallyacceptable and/or cosmeceutically acceptable vehicles.

Gels are semisolid, suspension-type systems. Single-phase gels containorganic macromolecules distributed substantially uniformly throughoutthe carrier liquid, which is typically aqueous, but also, preferably,contain an alcohol, and, optionally, an oil. Preferred “organicmacromolecules,” i.e., gelling agents, may be chemically crosslinkedpolymers such as crosslinked acrylic acid polymers, for instance, the“carbomer” family of polymers, e.g., carboxypolyalkylenes, that may beobtained commercially under the CARBOPOL® trademark. Also preferred incertain embodiments may be hydrophilic polymers such as polyethyleneoxides, polyoxyethylene-polyoxypropylene copolymers andpolyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropylmethylcellulose phthalate, and methyl cellulose; gums such as tragacanthand xanthan gum; sodium alginate; and gelatin. In order to prepare auniform gel, dispersing agents such as alcohol or glycerin can be added,or the gelling agent can be dispersed by trituration, mechanical mixingor stirring, or combinations thereof.

Ointments, as also well known in the art, are semisolid preparationsthat are typically based on petrolatum or other petroleum derivatives.The specific ointment base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for a number of desirablecharacteristics, e.g., emolliency or the like. As with other carriers orvehicles, an ointment base should be inert, stable, nonirritating, andnonsensitizing. As explained in Remington: The Science and Practice ofPharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages1399-1404, ointment bases may be grouped in four classes: oleaginousbases; emulsifiable bases; emulsion bases; and water-soluble bases.Oleaginous ointment bases include, for example, vegetable oils, fatsobtained from animals, and semisolid hydrocarbons obtained frompetroleum. Emulsifiable ointment bases, also known as absorbent ointmentbases, contain little or no water and include, for example,hydroxystearic sulfate, anhydrous lanolin, and hydrophilic petrolatum.Emulsion ointment bases are either water-in-oil (W/O) emulsions oroil-in-water (O/W) emulsions, and include, for example, cetyl alcohol,glyceryl monostearate, lanolin, and stearic acid. Preferredwater-soluble ointment bases are prepared from polyethylene glycols ofvarying molecular weight (see, e.g., Remington, Id.).

Pastes are semisolid dosage forms in which the active agent is suspendedin a suitable base. Depending on the nature of the base, pastes aredivided between fatty pastes or those made from single-phase aqueousgels. The base in a fatty paste is generally petrolatum or hydrophilicpetrolatum or the like. The pastes made from single-phase aqueous gelsgenerally incorporate carboxymethylcellulose or the like as a base.

Formulations may also be prepared with liposomes, micelles, andmicrospheres. Liposomes are microscopic vesicles having one(unilamellar) or a plurality (multilamellar) of lipid walls comprising alipid bilayer, and, in the present context, may encapsulate and/or haveadsorbed to their lipid membranous surfaces one or more components ofthe topical formulations herein described, such as the antiseptic, woundhealing/skin tissue/epithelial tissue repair-promoting compounds (e.g.,microparticulate BT compounds, optionally along with one or moreantibiotics) or certain carriers or excipients. Liposomal preparationsherein include cationic (positively charged), anionic (negativelycharged), and neutral preparations. Cationic liposomes are readilyavailable. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the tradename LIPOFECTIN® (GIBCO BRL, Grand Island,N.Y.). Similarly, anionic and neutral liposomes are readily available aswell, e.g., from Avanti Polar Lipids (Birmingham, Ala.), or can beeasily prepared using readily available materials. Such materialsinclude phosphatidyl choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), and dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with DOTMA in appropriate ratios. Methodsfor making liposomes using these materials are well known in the art.

Micelles are known in the art as comprised of surfactant moleculesarranged so that their polar headgroups form an outer spherical shell,while the hydrophobic, hydrocarbon chains are oriented towards thecenter of the sphere, forming a core. Micelles form in an aqueoussolution containing surfactant at a high enough concentration so thatmicelles naturally result. Surfactants useful for forming micellesinclude, but are not limited to, potassium laurate, sodium octanesulfonate, sodium decane sulfonate, sodium dodecane sulfonate, sodiumlauryl sulfate, docusate sodium, decyltrimethylammonium bromide,dodecyltriethylammonium bromide, tetradecyltrimethylammonium bromide,tetradecyltrimethyl-ammonium chloride, dodecylammonium chloride,polyoxyl-8dodecyl ether, polyoxyl-12 dodecyl ether, nonoxynol 10, andnonoxynol 30.

Microspheres, similarly, may be incorporated into the presentlydescribed topical formulations. Like liposomes and micelles,microspheres essentially encapsulate one or more components of thepresent formulations. They are generally, but not necessarily, formedfrom lipids, preferably charged lipids such as phospholipids.Preparation of lipidic microspheres is well known in the art.

Various additives, as known to those skilled in the art, may also beincluded in the topical formulations. For example, solvents, includingrelatively small amounts of alcohol, may be used to solubilize certainformulation components. It may be desirable, for certain topicalformulations or in cases of particularly severe skin injury such as apost-surgical acute or chronic wound or post-surgical dermal wounddehiscence, to include in the topical formulation an added skinpermeation enhancer in the formulation. Examples of suitable enhancersinclude, but are not limited to, ethers such as diethylene glycolmonoethyl ether (available commercially as sold under the trademarkTRANSCUTOL®) and diethylene glycol monomethyl ether; surfactants such assodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide,benzalkonium chloride, polyoxypropylene/polyoxyethylene copolymers knownas poloxamers (poloxamer 231, poloxamer 182, poloxamer 184) and soldunder the trademark PLURONIC® (BASF Corp.), polysorbate surfactants soldunder the trademark TWEEN® (TWEEN® 20, TWEEN® 40, TWEEN® 60, TWEEN® 80),and lecithin (U.S. Pat. No. 4,783,450); alcohols such as ethanol,propanol, octanol, benzyl alcohol, and the like; polyethylene glycol andesters thereof such as polyethylene glycol monolaurate (PEGML; see,e.g., U.S. Pat. No. 4,568,343); amides and other nitrogenous compoundssuch as urea, dimethylacetamide (DMA), dimethylformamide (DMF),2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine, andtriethanolamine; terpenes; alkanones; and organic acids, particularlycitric acid and succinic acid. The enhancer n-dodecylcaprolactam(laurocapram) sold under the trademark AZONE® and sulfoxides such asDMSO and C₁₀MSO may also be used, but are less preferred.

Most preferred skin permeation enhancers are those lipophilicco-enhancers typically referred to as “plasticizing” enhancers, i.e.,enhancers that have a molecular weight in the range of about 150 to 1000daltons, an aqueous solubility of less than about 1 wt %, preferablyless than about 0.5 wt %, and most preferably less than about 0.2 wt %.The Hildebrand solubility parameter of plasticizing enhancers is in therange of about 2.5 to about 10, preferably in the range of about 5 toabout 10. Preferred lipophilic enhancers are fatty esters, fattyalcohols, and fatty ethers. Examples of specific and most preferredfatty acid esters include methyl laurate, ethyl oleate, propylene glycolmonolaurate, propylene glycerol dilaurate, glycerol monolaurate,glycerol monooleate, isopropyl n-decanoate, and octyldodecyl myristate.Fatty alcohols include, for example, stearyl alcohol and oleyl alcohol,while fatty ethers include compounds wherein a diol or triol, preferablya C₂-C₄ alkane diol or triol, are substituted with one or two fattyether substituents. Additional skin permeation enhancers will be knownto those of ordinary skill in the art of topical drug delivery, and/orare described in the relevant literature. See, e.g., PercutaneousPenetration Enhancers, eds. Smith et al. (CRC Press, Boca Raton, Fla.,1995).

Various other additives may be included in the topical formulationsaccording to certain embodiments of the present invention, in additionto those identified above. These include, but are not limited to,antioxidants, astringents, perfumes, preservatives, emollients,pigments, dyes, humectants, propellants, and sunscreen agents, as wellas other classes of materials whose presence may be cosmetically,medicinally or otherwise desirable. Typical examples of optionaladditives for inclusion in the formulations of certain embodiments ofthe invention are as follows: preservatives such as sorbate; solventssuch as isopropanol and propylene glycol; astringents such as mentholand ethanol; emollients such as polyalkylene methyl glucosides;humectants such as glycerine; emulsifiers such as glycerol stearate,PEG-100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate60; sorbitol and other polyhydroxyalcohols such as polyethylene glycol;sunscreen agents such as octyl methoxyl cinnamate (availablecommercially as PARSOL® MCX) and butyl methoxy benzoylmethane (availableunder the tradename PARSOL® 1789); antioxidants such as ascorbic acid(vitamin C), α-tocopherol (Vitamin E), β-tocopherol, γ-tocopherol,δ-tocopherol, ε-tocopherol, ζ₁-tocopherol, ζ₂-tocopherol, η-tocopherol,and retinol (vitamin A); essential oils, ceramides, essential fattyacids, mineral oils, wetting agents and other surfactants such as thePLURONIC® series of hydrophilic polymers available from BASF (Mt. Olive,N.J.), vegetable oils (e.g., soy bean oil, palm oil, liquid fraction ofshea butter, sunflower oil), animal oils (e.g., perhydrosqualene),mineral oils, synthetic oils, silicone oils or waxes (e.g.,cyclomethicone and dimethicone), fluorinated oils (generallyperfluoropolyethers), fatty alcohols (e.g., cetyl alcohol), and waxes(e.g., beeswax, carnauba wax, and paraffin wax); skin-feel modifiers;and thickeners and structurants such as swelling clays and cross-linkedcarboxypolyalkylenes that may be obtained commercially under theCARBOPOL® trademark.

Other additives include beneficial agents such as those materials thatcondition the skin (particularly, the upper layers of the skin in thestratum corneum) and keep it soft by retarding the decrease of its watercontent and/or protect the skin. Such conditioners and moisturizingagents include, by way of example, pyrrolidine carboxylic acid and aminoacids; organic antimicrobial agents such as 2,4,4′-trichloro-2-hydroxydiphenyl ether (triclosan) and benzoic acid; anti-inflammatory agentssuch as acetylsalicylic acid and glycyrrhetinic acid; anti-seborrhoeicagents such as retinoic acid; vasodilators such as nicotinic acid;inhibitors of melanogenesis such as kojic acid; and mixtures thereof.Other advantageously included cosmeceutically active agents may bepresent, for example, α-hydroxyacids, α-ketoacids, polymerichydroxyacids, moisturizers, collagen, marine extracts, and antioxidantssuch as ascorbic acid (vitamin C), α-tocopherol (Vitamin E) or othertocopherols such as those described above, and retinol (vitamin A),and/or cosmetically acceptable salts, esters, amides, or otherderivatives thereof. Additional cosmetic agents include those that arecapable of improving oxygen supply in skin tissue, as described, forexample, in WO 94/00098 and WO 94/00109. Sunscreens may also beincluded.

Other embodiments may include a variety of non-carcinogenic,non-irritating healing materials that facilitate treatment with theformulations of certain embodiments of the invention. Such healingmaterials may include nutrients, minerals, vitamins, electrolytes,enzymes, herbs, plant extracts, honey, glandular or animal extracts, orsafe therapeutic agents that may be added to the formulation tofacilitate dermal healing. The amounts of these various additives arethose conventionally used in the cosmetics field, and range, forexample, from about 0.01% to about 20% of the total weight of thetopical formulation.

The formulations of certain embodiments of the invention may alsoinclude conventional additives such as opacifiers, fragrance, colorant,gelling agents, thickening agents, stabilizers, surfactants, and thelike. Other agents may also be added, such as antimicrobial agents, toprevent spoilage upon storage, i.e., to inhibit growth of microbes suchas yeasts and molds. Suitable antimicrobial agents are typicallyselected from methyl and propyl esters of p-hydroxybenzoic acid (e.g.,methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, andcombinations thereof. The formulations may also containirritation-mitigating additives to minimize or eliminate the possibilityof skin irritation or skin damage resulting from the anti-infectiveacute or chronic wound healing and skin tissue repair-promoting compoundto be administered, or from other components of the composition.Suitable irritation-mitigating additives include, for example:α-tocopherol; monoamine oxidase inhibitors, particularly phenyl alcoholssuch as 2-phenyl-1-ethanol; glycerin; salicylates; ascorbates;ionophores such as monensin; amphiphilic amines; ammonium chloride;N-acetylcysteine; capsaicin; and chloroquine. The irritation-mitigatingadditive, if present, may be incorporated into the topical formulationat a concentration effective to mitigate irritation or skin damage,typically representing not more than about 20 wt %, more typically notmore than about 5 wt %, of the formulation.

The topical formulations may also contain, in addition to theantiseptic/wound healing/anti-biofilm/skin tissue repair-promotingcompound (e.g., a BT compound, preferably as substantially homogeneousmicroparticles as provided herein, and optionally in combination withone or more synergizing antibiotics as described herein), atherapeutically effective amount of one or more additionalpharmacologically active agents suitable for topical administration.Such agents may include an asymmetrical lamellar aggregate consisting ofphospholipids and oxygen-loaded fluorocarbon or a fluorocarbon compoundmixture, which are capable of improving oxygen supply in skin tissue, asdescribed, for example, in International Patent Publication Nos. WO94/00098 and WO 94/00109.

Suitable pharmacologically active agents that may be incorporated intothe present topical formulations and thus topically applied, may includebut are not limited to, the following: agents that improve or eradicatepigmented or non-pigmented age spots, keratoses, and wrinkles;antimicrobial agents; antibacterial agents; antipruritic and antixeroticagents; antiinflammatory agents; local anesthetics and analgesics;corticosteroids; retinoids (e.g., retinoic acid; vitamins; hormones; andantimetabolites. Some examples of topical pharmacologically activeagents include acyclovir, amphotericins, chlorhexidine, clotrimazole,ketoconazole, econazole, miconazole, metronidazole, minocycline,nystatin, neomycin, kanamycin, phenyloin, para-amino benzoic acidesters, octyl methoxycinnamate, octyl salicylate, oxybenzone,dioxybenzone, tocopherol, tocopheryl acetate, selenium sulfide, zincpyrithione, diphenhydramine, pramoxine, lidocaine, procaine,erythromycin, tetracycline, clindamycin, chromatin, hydroquinone and itsmonomethyl and benzyl ethers, naproxen, ibuprofen, cromolyn, retinoicacid, retinol, retinyl palmitate, retinyl acetate, coal tar,griseofulvin, estradiol, hydrocortisone, hydrocortisone 21-acetate,hydrocortisone 17-valerate, hydrocortisone 17-butyrate, progesterone,betamethasone valerate, betamethasone dipropionate, triamcinoloneacetonide, fluocinonide, clobetasol propionate, minoxidil, dipyridamole,diphenylhydantoin, benzoyl peroxide, and 5-fluorouracil. As also notedabove, certain embodiments contemplate inclusion in the formulation ofan antibiotic such as a carbapenem antibiotic, a cephalosporinantibiotic, a fluoroquinolone antibiotic, a glycopeptide antibiotic, alincosamide antibiotic, a penicillinase-resistant penicillin antibiotic,an aminopenicillin antibiotic, or an aminoglycoside antibiotic such asamikacin.

A pharmacologically acceptable carrier may also be incorporated in thetopical formulation of certain present embodiments and may be anycarrier conventionally used in the art. Examples include water, loweralcohols, higher alcohols, honey, polyhydric alcohols, monosaccharides,disaccharides, polysaccharides, sugar alcohols such as, for example,glycols (2-carbon), glycerols (3-carbon), erythritols and threitols(4-carbon), arabitols, xylitols and ribitols (5-carbon), mannitols,sorbitols, dulcitols and iditols (6-carbon), isomaltols, maltitols,lactitols and polyglycitols, hydrocarbon oils, fats and oils, waxes,fatty acids, silicone oils, nonionic surfactants, ionic surfactants,silicone surfactants, and water-based mixtures and emulsion-basedmixtures of such carriers.

Topical formulation embodiments of the present invention may be appliedregularly to whatever acute or chronic wound site (e.g., the wounditself and surrounding tissue, including surrounding tissue that appearsunaffected by infection or otherwise normal or healthy) or skin area orother epithelial tissue surface (e.g., gastrointestinal tract,respiratory tract, glandular tissue) requires treatment with thefrequency and in the amount necessary to achieve the desired results.The frequency of treatment depends on the nature of the skin (or otherepithelial tissue) condition (e.g., an acute or chronic wound or otherskin wound such as may be found in dehiscence that results from asurgical incision, or other types of skin wounds), the degree of damageor deterioration of the skin (or other tissue), the responsiveness ofthe user's skin (or other tissue), the strength of the activeingredients (e.g., the herein describedwound-healing/antiseptic/anti-biofilm/skin tissue repair-promotingcompounds such as a BT compound and optionally one or more additionalpharmaceutically active ingredients, such as an antibiotic, e.g.,amikacin or other antibiotic) in the particular embodiment, theeffectiveness of the vehicle used to deliver the active ingredients intothe appropriate layer of the skin (or other epithelialsurface-containing tissue), the ease with which the formula is removedby physical contact with bandages or other dressings or clothing, or itsremoval by sweat or other intrinsic or extrinsic fluids, and theconvenience to the subject's or patient's activity level or lifestyle.

Typical concentrations of active substances such as the BT compoundantiseptic/anti-biofilm/wound-healing/skin tissue repair-promotingcompositions described herein can range, for example, from about0.001-30% by weight based on the total weight of the composition, toabout 0.01-5.0%, and more preferably to about 0.1-2.0%. As onerepresentative example, compositions of these embodiments of the presentinvention may be applied to an acute or chronic wound and/or to the skinat a rate equal to from about 1.0 mg/cm² of skin to about 20.0 mg/cm² ofskin. Representative examples of topical formulations include, but arenot limited to, aerosols, alcohols, anhydrous bases (such as lipsticksand powders), aqeuous solutions, creams, emulsions (including eitherwater-in-oil or oil-in-water emulsions), fats, foams, gels,hydro-alcoholic solutions, liposomes, lotions, microemulsions,ointments, oils, organic solvents, polyols, polymers, powders, salts,silicone derivatives, and waxes. Topical formulations may include, forexample, chelating agents, conditioning agents, emollients, excipients,humectants, protective agents, thickening agents, or UV absorbingagents. One skilled in the art will appreciate that formulations otherthan those listed may be used in embodiments of the present invention.

Chelating agents may be optionally included in topical formulations, andmay be selected from any agent that is suitable for use in a cosmeticcomposition, and may include any natural or synthetic chemical which hasthe ability to bind divalent cationic metals such as Ca²⁺, Mn²⁺, orMg²⁺. Examples of chelating agents include, but are not limited to EDTA,disodium EDTA, EGTA, citric acid, and dicarboxylic acids.

Conditioning agents may also be optionally included in topicalformulations. Examples of skin conditioning agents include, but are notlimited to, acetyl cysteine, N-acetyl dihydrosphingosine,acrylates/behenyl acrylate/dimethicone acrylate copolymer, adenosine,adenosine cyclic phosphate, adenosine phosphate, adenosine triphosphate,alanine, albumen, algae extract, allantoin and deriviatives, aloebarbadensis extracts, aluminum PCA, amyloglucosidase, arbutin, arginine,azulene, bromelain, buttermilk powder, butylene glycol, caffeine,calcium gluconate, capsaicin, carbocysteine, carnosine, beta-carotene,casein, catalase, cephalins, ceramides, chamomilla recutita (matricaria)flower extract, cholecalciferol, cholesteryl esters, coco-betaine,coenzyme A, corn starch modified, crystallins, cycloethoxymethicone,cysteine DNA, cytochrome C, darutoside, dextran sulfate, dimethiconecopolyols, dimethylsilanol hyaluronate, DNA, elastin, elastin aminoacids, epidermal growth factor, ergocalciferol, ergosterol, ethylhexylPCA, fibronectin, folic acid, gelatin, gliadin, beta-glucan, glucose,glycine, glycogen, glycolipids, glycoproteins, glycosaminoglycans,glycosphingolipids, horseradish peroxidase, hydrogenated proteins,hydrolyzed proteins, jojoba oil, keratin, keratin amino acids, andkinetin, lactoferrin, lanosterol, lauryl PCA, lecithin, linoleic acid,linolenic acid, lipase, lysine, lysozyme, malt extract, maltodextrin,melanin, methionine, mineral salts, niacin, niacinamide, oat aminoacids, oryzanol, palmitoyl hydrolyzed proteins, pancreatin, papain, PEG,pepsin, phospholipids, phytosterols, placental enzymes, placentallipids, pyridoxal 5-phosphate, quercetin, resorcinol acetate,riboflavin, RNA, saccharomyces lysate extract, silk amino acids,sphingolipids, stearamidopropyl betaine, stearyl palmitate, tocopherol,tocopheryl acetate, tocopheryl linoleate, ubiquinone, vitis vinifera(grape) seed oil, wheat amino acids, xanthan gum, and zinc gluconate.Skin conditioning agents other than those listed above may be combinedwith a disclosed composition or preparation provided thereby, as can bereadily appreciated by one skilled in the art.

Topical formulations may also optionally include one or more emollients,examples of which include, but are not limited to, acetylated lanolin,acetylated lanolin alcohol, acrylates/C₁₀₋₃₀ alkyl acrylatecrosspolymer, acrylates copolymer, alanine, algae extract, aloebarbadensis extract or gel, althea officinalis extract, aluminum starchoctenylsuccinate, aluminum stearate, apricot (prunus armeniaca) kerneloil, arginine, arginine aspartate, arnica montana extract, ascorbicacid, ascorbyl palmitate, aspartic acid, avocado (persea gratissima)oil, barium sulfate, barrier sphingolipids, butyl alcohol, beeswax,behenyl alcohol, beta-sitosterol, BHT, birch (betula alba) bark extract,borage (borago officinalis) extract, 2-bromo-2-nitropropane-1,3-diol,butcherbroom (ruscus aculeatus) extract, butylene glycol, calendulaofficinalis extract, calendula officinalis oil, candelilla (euphorbiacerifera) wax, canola oil, caprylic/capric triglyceride, cardamon(elettaria cardamomum) oil, carnauba (copernicia cerifera) wax,carrageenan (chondrus crispus), carrot (daucus carota sativa) oil,castor (ricinus communis) oil, ceramides, ceresin, ceteareth-5,ceteareth-12, ceteareth-20, cetearyl octanoate, ceteth-20, ceteth-24,cetyl acetate, cetyl octanoate, cetyl palmitate, chamomile (anthemisnobilis) oil, cholesterol, cholesterol esters, cholesterylhydroxystearate, citric acid, clary (salvia sclarea) oil, cocoa(theobroma cacao) butter, coco-caprylate/caprate, coconut (cocosnucifera) oil, collagen, collagen amino acids, corn (zea mays) oil,fatty acids, decyl oleate, dextrin, diazolidinyl urea, dimethiconecopolyol, dimethiconol, dioctyl adipate, dioctyl succinate,dipentaerythrityl hexacaprylate/hexacaprate, DMDM hydantoin, DNA,erythritol, ethoxydiglycol, ethyl linoleate, eucalyptus globulus oil,evening primrose (oenothera biennis) oil, fatty acids, tructose,gelatin, geranium maculatum oil, glucosamine, glucose glutamate,glutamic acid, glycereth-26, glycerin, glycerol, glyceryl distearate,glyceryl hydroxystearate, glyceryl laurate, glyceryl linoleate, glycerylmyristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE,glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape(vitis vinifera) seed oil, hazel (corylus americana) nut oil, hazel(corylus avellana) nut oil, hexylene glycol, honey, hyaluronic acid,hybrid safflower (carthamus tinctorius) oil, hydrogenated castor oil,hydrogenated coco-glycerides, hydrogenated coconut oil, hydrogenatedlanolin, hydrogenated lecithin, hydrogenated palm glyceride,hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenatedtallow glyceride, hydrogenated vegetable oil, hydrolyzed collagen,hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin,hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline,imidazolidinyl urea, iodopropynyl butylcarbamate, isocetyl stearate,isocetyl stearoyl stearate, isodecyl oleate, isopropyl isostearate,isopropyl lanolate, isopropyl myristate, isopropyl palmitate, isopropylstearate, isostearamide DEA, isostearic acid, isostearyl lactate,isostearyl neopentanoate, jasmine (jasminum officinale) oil, jojoba(buxus chinensis) oil, kelp, kukui (aleurites moluccana) nut oil,lactamide MEA, laneth-16, laneth-10 acetate, lanolin, lanolin acid,lanolin alcohol, lanolin oil, lanolin wax, lavender (lavandulaangustifolia) oil, lecithin, lemon (citrus medica limonum) oil, linoleicacid, linolenic acid, macadamia ternifolia nut oil, magnesium stearate,magnesium sulfate, maltitol, matricaria (chamomilla recutita) oil,methyl glucose sesquistearate, methylsilanol PCA, microcrystalline wax,mineral oil, mink oil, mortierella oil, myristyl lactate, myristylmyristate, myristyl propionate, neopentyl glycol dicaprylate/dicaprate,octyldodecanol, octyldodecyl myristate, octyldodecyl stearoyl stearate,octyl hydroxystearate, octyl palmitate, octyl salicylate, octylstearate, oleic acid, olive (olea europaea) oil, orange (citrusaurantium dulcis) oil, palm (elaeis guineensis) oil, palmitic acid,pantethine, panthenol, panthenyl ethyl ether, paraffin, PCA, peach(prunus persica) kernel oil, peanut (arachis hypogaea) oil, PEG-8 C12 18ester, PEG-15 cocamine, PEG-150 distearate, PEG-60 glyceryl isostearate,PEG-5 glyceryl stearate, PEG-30 glyceryl stearate, PEG-7 hydrogenatedcastor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castoroil, PEG-20 methyl glucose sesquistearate, PEG-40 sorbitan peroleate,PEG-5 soy sterol, PEG-10 soy sterol, PEG-2 stearate, PEG-8 stearate,PEG-20 stearate, PEG-32 stearate, PEG-40 stearate, PEG-50 stearate,PEG-100 stearate, PEG-150 stearate, pentadecalactone, peppermint (menthapiperita) oil, petrolatum, phospholipids, polyamino sugar condensate,polyglyceryl-3 diisostearate, polyquaternium-24, polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85,potassium myristate, potassium palmitate, potassium sorbate, potassiumstearate, propylene glycol, propylene glycol dicaprylate/dicaprate,propylene glycol dioctanoate, propylene glycol dipelargonate, propyleneglycol laurate, propylene glycol stearate, propylene glycol stearate SE,PVP, pyridoxine dipalmitate, quaternium-15, quaternium-18 hectorite,quaternium-22, retinol, retinyl palmitate, rice (oryza sativa) bran oil,RNA, rosemary (rosmarinus officinalis) oil, rose oil, safflower(carthamus tinctorius) oil, sage (salvia officinalis) oil, salicylicacid, sandalwood (santalum album) oil, serine, serum protein, sesame(sesamum indicum) oil, shea butter (butyrospermum parkii), silk powder,sodium chondroitin sulfate, sodium DNA, sodium hyaluronate, sodiumlactate, sodium palmitate, sodium PCA, sodium polyglutamate, sodiumstearate, soluble collagen, sorbic acid, sorbitan laurate, sorbitanoleate, sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate,sorbitol, soybean (glycine soja) oil, sphingolipids, squalane, squalene,stearamide MEA-stearate, stearic acid, stearoxy dimethicone,stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate,stearyl heptanoate, stearyl stearate, sunflower (helianthus annuus) seedoil, sweet almond (prunus amygdalus dulcis) oil, synthetic beeswax,tocopherol, tocopheryl acetate, tocopheryl linoleate, tribehenin,tridecyl neopentanoate, tridecyl stearate, triethanolamine, tristearin,urea, vegetable oil, water, waxes, wheat (triticum vulgare) germ oil,and ylang ylang (cananga odorata) oil.

In some embodiments a topical formulation may contain a suitableexcipient, which typically should have a high affinity for the skin, bewell tolerated, stable, and yield a consistency that allows for easyutilization. Suitable topical excipients and vehicles can be routinelyselected for a particular use by those skilled in the art, andespecially with reference to one of many standard texts in the art, suchas Remington's Pharmaceutical Sciences, Vol. 18, Mack Publishing Co.,Easton, Pa. (1990), in particular Chapter 87. Optionally one or morehumectants are also included in the topical formulation. Examples ofhumectants include, but are not limited to, amino acids, chondroitinsulfate, diglycerin, erythritol, fructose, glucose, glycerin, glycerol,glycol, 1,2,6-hexanetriol, honey, hyaluronic acid, hydrogenated honey,hydrogenated starch hydrolysate, inositol, lactitol, maltitol, maltose,mannitol, natural moisturization factor, PEG-15 butanediol, polyglycerylsorbitol, salts of pyrollidone carboxylic acid, potassium PCA, propyleneglycol, sodium glucuronate, sodium PCA, sorbitol, sucrose, trehalose,urea, and xylitol.

Certain embodiments contemplate topical formulations containing one ormore additional skin protective agent. Examples of skin protectiveagents may include, but are not limited to, algae extract, allantoin,aluminum hydroxide, aluminum sulfate, betaine, camellia sinensis leafextract, cerebrosides, dimethicone, glucuronolactone, glycerin, kaolin,lanolin, malt extract, mineral oil, petrolatum, potassium gluconate, andtalc. One skilled in the art will readily appreciate that skinprotectants other than those listed above may also be combined with adisclosed composition of the present invention or preparation providedthereby.

Surfactants may also desirably be included in certain topicalformulations contemplated herein, and can be selected from any naturalor synthetic surfactants suitable for use in cosmetic compositions, suchas cationic, anionic, zwitterionic, or non-ionic surfactants, ormixtures thereof. (See Rosen, M., “Surfactants and InterfacialPhenomena,” Second Edition, John Wiley & Sons, New York, 1988, Chapter1, pages 4 31). Examples of cationic surfactants may include, but arenot limited to, DMDAO or other amine oxides, long-chain primary amines,diamines and polyamines and their salts, quaternary ammonium salts,polyoxyethylenated long-chain amines, and quaternized polyoxyethylenatedlong-chain amines. Examples of anionic surfactants may include, but arenot limited to, SDS; salts of carboxylic acids (e.g., soaps); salts ofsulfonic acids, salts of sulfuric acid, phosphoric and polyphosphoricacid esters; alkylphosphates; monoalkyl phosphate (MAP); and salts ofperfluorocarboxylic acids. Examples of zwitterionic surfactants mayinclude, but are not limited to, cocoamidopropyl hydroxysultaine (CAPHS)and others which are pH-sensitive and require special care in designingthe appropriate pH of the formula (I.e., alkylaminopropionic acids,imidazoline carboxylates, and betaines) or those which are notpH-sensitive (e.g., sulfobetaines, sultaines). Examples of non-ionicsurfactants may include, but are not limited to, alkylphenolethoxylates, alcohol ethoxylates, polyoxyethylenated polyoxypropyleneglycols, polyoxyethylenated mercaptans, long-chain carboxylic acidesters, alkanolamides, tertiary acetylenic glycols, polyoxyethylenatedsilicones, N-alkypyrrolidones, and alkylpolyglycosidases. Wettingagents, mineral oil or other surfactants such as non-ionic detergents oragents such as one or more members of the PLURONICS® series (BASF, Mt.Olive, N.J.) may also be included, for example and according tonon-limiting theory, to discourage aggregation of BT microparticleswithin the microparticulate suspension. Any combination of surfactantsis acceptable. Certain embodiments may include at least one anionic andone cationic surfactant, or at least one cationic and one zwitterionicsurfactant which are compatible, i.e., do not form complexes whichprecipitate appreciably when mixed.

Examples of thickening agents that may also be present in certaintopical formulations include, but are not limited to, acrylamidescopolymer, agarose, amylopectin, bentonite, calcium alginate, calciumcarboxymethyl cellulose, carbomer, carboxymethyl chitin, cellulose gum,dextrin, gelatin, hydrogenated tallow, hydroxytheylcellulose,hydroxypropylcellulose, hydroxypropyl starch, magnesium alginate,methylcellulose, microcrystalline cellulose, pectin, various PEG's,polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, variousPPG's, sodium acrylates copolymer, sodium carrageenan, xanthan gum, andyeast beta-glucan. Thickening agents other than those listed above mayalso be used in embodiments of this invention.

According to certain embodiments contemplated herein, a topicalformulation may comprise one or more sunscreening or UV absorbingagents. Where ultraviolet light- (UVA and UVB) absorbing properties aredesired, such agents may include, for example, benzophenone,benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-4,benzophenone-5, benzophenone-6, benzophenone-7, benzophenone-8,benzophenone-9, benzophenone-10, benzophenone-11, benzophenone-12,benzyl salicylate, butyl PABA, cinnamate esters, cinoxate,DEA-methoxycinnamate, diisopropyl methyl cinnamate, ethyldihydroxypropyl PABA, ethyl diisopropylcinnamate, ethylmethoxycinnamate, ethyl PABA, ethyl urocanate, glyceryl octanoatedimethoxycinnamate, glyceryl PABA, glycol salicylate, homosalate,isoamyl p-methoxycinnamate, oxides of titanium, zinc, zirconium,silicon, manganese, and cerium, PABA, PABA esters, butyl methoxybenzoylmethane (available under the tradename PARSOL® 1789), andisopropylbenzyl salicylate, and mixtures thereof. One skilled in the artwill appreciate that sunscreening and UV absorbing or protective agentsother than those listed may be used in certain embodiments of thepresent invention.

Topical formulations disclosed herein are typically effective at pHvalues between about 2.5 and about 10.0. Preferably, the pH of thecomposition is at or about the following pH ranges: about pH 5.5 toabout pH 8.5, about pH 5 to about pH 10, about pH 5 to about pH 9, aboutpH 5 to about pH 8, about pH 3 to about pH 10, about pH 3 to about pH 9,about pH 3 to about pH 8, and about pH 3 to about pH 8.5. Mostpreferably, the pH is about pH 7 to about pH 8. One of ordinary skill inthe art may add appropriate pH adjusting ingredients to the compositionsof the present invention to adjust the pH to an acceptable range.“About” a specified pH is understood by those familiar with the art toinclude formulations in which at any given time the actual measured pHmay be less or more than the specified value by no more than 0.7, 0.6,0.5, 0.4, 0.3, 0.2 or 0.1 pH units, where it is recognized thatformulation composition and storage conditions may result in drifting ofpH from an original value.

A cream, lotion, gel, ointment, paste or the like may be spread on theaffected surface and gently rubbed in. A solution may be applied in thesame way, but more typically will be applied with a dropper, swab, orthe like, and carefully applied to the affected areas. The applicationregimen will depend on a number of factors that may readily bedetermined, such as the severity of the wound and its responsiveness toinitial treatment, but will normally involve one or more applicationsper day on an ongoing basis. One of ordinary skill may readily determinethe optimum amount of the formulation to be administered, administrationmethodologies and repetition rates. In general, it is contemplated thatthe formulations of these and related embodiments of the invention willbe applied in the range of once or twice or more weekly up to once,twice, thrice, four times or more daily.

As also discussed above, the topical formulations useful herein thusalso contain a pharmaceutically acceptable carrier, including anysuitable diluent or excipient, which includes any pharmaceutical agentthat does not itself harm the subject receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable carriers include, but are not limited to, liquids, such aswater, saline, glycerol and ethanol, and the like, and may also includeviscosity enhancers (e.g., balsam fir resin) or film-formers such ascollodion or nitrocellulose solutions. A thorough discussion ofpharmaceutically acceptable carriers, diluents, and other excipients ispresented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J.current edition).

When the topical formulation is in the form of a gel- or liquid-filledcapsule, for example, a gelatin capsule, it may contain, in addition tomaterials of the above type, a liquid carrier such as polyethyleneglycol or oil. The liquid pharmaceutical compositions of certainembodiments of the invention, whether they be solutions, suspensions orother like form, may include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or diglycerides which may serve as thesolvent or suspending medium, polyethylene glycols, glycerin, propyleneglycol or other solvents; antibacterial agents such as benzyl alcohol ormethyl paraben; additional antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose.

For topical administration the carrier may suitably comprise a solution,emulsion, ointment or gel base. The base, for example, may comprise oneor more of the following: petrolatum, lanolin, polyethylene glycols, beewax, mineral oil, diluents such as water and alcohol, and emulsifiersand stabilizers. Thickening agents may be present in a pharmaceutical orcosmeceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the compound of certain embodiments of the inventionfrom about 0.1 to about 10% w/v (weight per unit volume). A topicalformulation may be provided in the form of a cream, lotion, solution,spray, gel, ointment, paste or the like, and/or may contain liposomes,micelles, microspheres and/or other microparticle or nanoparticledelivery elements. A topical formulation may also be provided in theform of time-release or sustained release particles or pellets, forexample, slow-release ethylene vinyl acetate polymer (e.g., ELVAX®40,Aldrich, Milwaukee, Wis.) pellets, that can be directly administered toa wound site.

The topical formulation may include an agent that binds to the skintissue repair-promoting compound and thereby assists in its delivery toskin epithelial cells (e.g., keratinocytes) and/or fibroblasts. Suitableagents that may act in this capacity include clathrating agents such ascyclodextrins; other agents may include a protein or a liposome.

The topical formulation of certain embodiments of the invention may alsobe provided in the form of dosage units that can be administered as anaerosol. The term aerosol is used to denote a variety of systems rangingfrom those of colloidal nature to systems consisting of pressurizedpackages. Delivery may be by a liquefied or compressed gas or by asuitable pump system that dispenses the active ingredients. Aerosols ofcompounds of certain embodiments of the invention may be delivered insingle phase, bi-phasic, or tri-phasic systems in order to deliver theactive ingredient(s). Delivery of the aerosol includes the necessarycontainer, activators, valves, subcontainers, and the like, whichtogether may form a kit. One skilled in the art, without undueexperimentation may determine preferred aerosols for delivering topicalformulations to the skin or to a wound site.

The topical formulations may be prepared by methodology well known inthe pharmaceutical art. For example, a pharmaceutical compositionintended to be administered to a wound site or to the skin as a spray,wash or rinse can be prepared by combining a BTantiseptic/wound-healing/anti-biofilm/skin tissue repair-promotingcompound as described herein with sterile, distilled water so as to forma solution. A surfactant may be added to facilitate the formation of ahomogeneous solution or suspension. Surfactants are compounds thatnon-covalently interact with the antioxidant active compound so as tofacilitate dissolution or homogeneous suspension of the compound in theaqueous delivery system.

The BT antiseptic/wound-healing/anti-biofilm/skin tissuerepair-promoting compounds for use in topical formulations, or theirpharmaceutically acceptable salts, are administered in a therapeuticallyeffective amount, which will vary depending upon a variety of factorsincluding the nature of the wound site (where relevant), the activity ofthe specific BT compound employed (including the inclusion or absencefrom the formulation of an antibiotic, such as an aminoglycosideantibiotic, e.g., amikacin); the metabolic stability and length ofaction of the compound; the age, body weight, general health, sex, skintype, immune status and diet of the subject; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular skin wound for which skin tissue repair isdesired; and the subject undergoing therapy. Generally, atherapeutically effective daily dose is (for a 70 kg mammal) from about0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferablya therapeutically effective dose is (for a 70 kg mammal) from about 0.01mg/kg (i.e., 7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably atherapeutically effective dose is (for a 70 kg mammal) from about 1mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g).

The ranges of effective doses provided herein are not intended to belimiting and represent preferred dose ranges. However, the mostpreferred dosage will be tailored to the individual subject, as isunderstood and determinable by one skilled in the relevant arts. (see,e.g., Berkow et al., eds., The Merck Manual, 16^(th) edition, Merck andCo., Rahway, N.J., 1992; Goodman et al., eds., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10^(th) edition, Pergamon Press,Inc., Elmsford, N.Y., (2001); Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, Ltd., Williams and Wilkins, Baltimore, Md. (1987); Ebadi,Pharmacology, Little, Brown and Co., Boston, (1985); Osolci al., eds.,Remington's Pharmaceutical Sciences, 18^(th) edition, Mack PublishingCo., Easton, Pa. (1990); Katzung, Basic and Clinical Pharmacology,Appleton and Lange, Norwalk, Conn. (1992)).

The total dose required for each treatment can be administered bymultiple doses or in a single dose over the course of the day, ifdesired. Certain preferred embodiments contemplate a single applicationof the topical formulation per day. Generally, and in distinctembodiments, treatment may be initiated with smaller dosages, which areless than the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached.

The topical formulation can be administered alone or in conjunction withother treatments and/or pharmaceuticals directed to the skin wound, ordirected to other associated symptoms or etiologic factors. For example,and as also noted above, the topical formulation may further compriseretinoic acid. As another example, the topical formulation may compriseone or more skin tissue repair-promoting compounds described herein, ormay comprise two or more such compounds having different cellular woundrepair activities.

The recipients of the topical formulations described herein can be anyvertebrate animal, such as mammals. Among mammals, the preferredrecipients are mammals of the Orders Primate (including humans, apes andmonkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs),Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora(including cats, and dogs). Among birds, the preferred recipients areturkeys, chickens and other members of the same order. The mostpreferred recipients are humans, and particularly preferred are humanshaving one or more acute or chronic wounds or wounds that containbiofilms.

For topical applications, it is preferred to administer an effectiveamount of a pharmaceutical composition comprising a BT compoundantiseptic/wound-healing/anti-biofilm/skin tissue repair-promotingcompound according to the herein described embodiments, to a targetarea, e.g., a skin wound such as an acute or chronic wound, and/or anat-risk area (e.g., for wound dehiscence) of the skin, and the like.This amount will generally range from about 0.0001 mg to about 1 g of acompound of certain embodiments of the invention per application,depending upon the area to be treated, the severity of the wound (or ofa past or contemplated surgical incision), and the nature of the topicalvehicle employed. A preferred topical preparation is an ointment orslow-release pellets, wherein about 0.001 to about 50 mg of activeingredient is used per cc of ointment base or pellet suspension. Thepharmaceutical composition can be formulated as transdermal compositionsor transdermal delivery devices (“patches”). Such compositions include,for example, a backing, active compound reservoir, a control membrane,liner and contact adhesive. Such transdermal patches may be used toprovide continuous pulsatile, or on demand delivery of the compounds ofthe present invention as desired.

The compositions of certain embodiments can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures known in theart. Controlled release drug delivery systems include osmotic pumpsystems and dissolutional systems containing polymer-coated reservoirsor drug-polymer matrix formulations. Examples of controlled releasesystems are given in U.S. Pat. Nos. 3,845,770 and 4,326,525 and in P. J.Kuzma et al, Regional Anesthesia 22 (6): 543-551 (1997), all of whichare incorporated herein by reference.

The most suitable route will depend on the nature and severity of thecondition being treated. Those skilled in the art are also familiar withdetermining topical administration methods (sprays, creams, openapplication, occlusive dressing, soaks, washes, etc.), dosage forms,suitable pharmaceutical excipients and other matters relevant to thedelivery of the compounds to a subject in need thereof.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are required and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural references unless the content clearlydictates otherwise. As used herein, in particular embodiments, the terms“about” or “approximately” when preceding a numerical value indicatesthe value plus or minus a range of 5%, 6%, 7%, 8% or 9%. In otherembodiments, the terms “about” or “approximately” when preceding anumerical value indicates the value plus or minus a range of 10%, 11%,12%, 13% or 14%. In yet other embodiments, the terms “about” or“approximately” when preceding a numerical value indicates the valueplus or minus a range of 15%, 16%, 17%, 18%, 19% or 20%.

The following Examples are presented by way of illustration and notlimitation.

EXAMPLES Example 1 Preparation of BT Compounds

The following BT compounds were prepared either according to the methodsof Domenico et al. (U.S. RE37,793, U.S. Pat. No. 6,248,371, U.S. Pat.No. 6,086,921, U.S. Pat. No. 6,380,248) or as microparticles accordingto the synthetic protocol described below for BisEDT. Shown are atomicratios relative to a single bismuth atom, for comparison, based on thestoichiometric ratios of the reactants used and the known propensity ofbismuth to form trivalent complexes with sulfur containing compounds.The numbers in parenthesis are the ratios of bismuth to one (or more)thiol agents (e.g. Bi:thiol1/thiol2; see also Table 1).

1) CPD 1B-1 Bis-EDT (1:1) BiC2H4S2

2) CPD 1B-2 Bis-EDT (1:1.5) BiC₃H₆S₃

3) CPD 1B-3 Bis-EDT (1:1.5) BiC₃H₆S₃

4) CPD 1C Bis-EDT (soluble Bi prep.) (1:1.5) BiC₃H₆S₃

5) CPD 2A Bis-Bal (1:1) BiC₃H₆S₂O

6) CPD 2B Bis-Bal (1:1.5) BiC_(4.5)H₉O_(1.5)S₃

7) CPD 3A Bis-Pyr (1:1.5) BiC_(7.5)H₆N_(1.5)O_(1.5)S_(1.5)

8) CPD 3B Bis-Pyr (1:3) BiC₁₅H₁₂N₃O₃S₃

9) CPD 4 Bis-Ery (1:1.5) BiC₆H₁₂O₃S₃

10) CPD 5 Bis-Tol (1:1.5) BiC_(10.5)H₉S₃

11) CPD 6 Bis-BDT (1:1.5) BiC₆H₁₂S₃

12) CPD 7 Bis-PDT (1:1.5) BiC_(4.5)H₉S₃

13) CPD 8-1 Bis-Pyr/BDT (1:1/1)

14) CPD 8-2 Bis-Pyr/BDT (1:1/0.5)

15) CPD 9 Bis-2hydroxy, propane thiol (1:3)

16) CPD 10 Bis-Pyr/Bal (1:1/0.5)

17) CPD 11 Bis-Pyr/EDT (1:1/0.5)

18) CPD 12 Bis-Pyr/Tol (1:1/0.5)

19) CPD 13 Bis-Pyr/PDT (1:1/0.5)

20) CPD 14 Bis-Pyr/Ery (1:1/0.5)

21) CPD 15 Bis-EDT/2hydroxy, propane thiol (1:1/1)

Microparticulate bismuth-1,2-ethanedithiol (Bis-EDT, soluble bismuthpreparation) was prepared as follows:

To an excess (11.4 L) of 5% aqueous HNO₃ at room temperature in a 15 Lpolypropylene carboy was slowly added by dropwise addition 0.331 L(˜0.575 moles) of an aqueous Bi(NO₃)₃ solution (43% Bi(NO₃)₃ (w/w), 5%nitric acid (w/w), 52% water (w/w), Shepherd Chemical Co., Cincinnati,Ohio, product no. 2362; δ˜1.6 g/mL) with stirring, followed by slowaddition of absolute ethanol (4 L). Some white precipitate formed butwas dissolved by continued stirring. An ethanolic solution (˜1.56 L,˜0.55 M) of 1,2-ethanedithiol (CAS 540-63-6) was separately prepared byadding, to 1.5 L of absolute ethanol, 72.19 mL (0.863 moles) of1,2-ethanedithiol using a 60 mL syringe, and then stirring for fiveminutes. The 1,2-ethanedithiol/EtOH reagent was then slowly added bydropwise addition over the course of five hours to the aqueousBi(NO₃)₃/HNO₃ solution, with continued stirring overnight. The formedproduct was allowed to settle as a precipitate for approximately 15minutes, after which the filtrate was removed at 300 mL/min using aperistaltic pump. The product was then collected by filtration on finefilter paper in a 15-cm diameter Buchner funnel, and washed sequentiallywith three, 500-mL volumes each of ethanol, USP water, and acetone toobtain BisEDT (694.51 gm/mole) as a yellow amorphous powdered solid. Theproduct was placed in a 500 mL amber glass bottle and dried over CaCl₂under high vacuum for 48 hours. Recovered material (yield ˜200 g) gaveoff a thiol-characteristic odor. The crude product was redissolved in750 mL of absolute ethanol, stirred for 30 min, then filtered and washedsequentially with 3×50 mL ethanol, 2×50 mL acetone, and washed againwith 500 mL of acetone. The rewashed powder was triturated in 1M NaOH(500 mL), filtered and washed with 3×220 mL water, 2×50 mL ethanol, and1×400 mL acetone to afford 156.74 gm of purified BisEDT. Subsequentbatches prepared in essentially the same manner resulted in yields ofabout 78-91%.

The product was characterized as having the structure shown above informula I by analysis of data from ¹H and ¹³C nuclear magnetic resonance(NMR), infrared spectroscopy (IR), ultraviolet spectroscopy (UV), massspectrometry (MS) and elemental analysis. An HPLC method was developedto determine chemical purity of BisEDT whereby the sample was preparedin DMSO (0.5 mg/mL). The λ_(max) was determined by scanning a solutionof BisEDT in DMSO between 190 and 600 nm. Isocratic HPLC elution at 1mL/min was performed at ambient temperature in a mobile phase of 0.1%formic acid in acetonitrile:water (9:1) on a WATERS® (Millipore Corp.,Milford, Mass.) model 2695 chromatograph with UV detector monitoring at265 nm (λ_(max)), 2 μL injection volume, equipped with a YMC-PACK® PVCSil NP, 5 μm, 250×4.6 mm inner diameter analytical column (WATERS®) anda single peak was detected, reflecting chemical purity of 100±0.1%.Elemental analysis was consistent with the structure of formula (I).

The dried particulate matter was characterized to assess the particlesize properties. Briefly, microparticles were resuspended in 2%polyoxypropylene/polyoxyethylene copolymers sold under the trademarkPLURONIC® F-68 (BASF, Mt. Olive, N.J.) and the suspension was sonicatedfor 10 minutes in a water bath sonicator at standard setting prior toanalysis using a NANOSIZER™/ZETASIZER® Nano-S particle analyzer (modelZEN1600 (without zeta-potential measuring capacity), MALVERNINSTRUMENTS™, Worcestershire, UK) according to the manufacturer'srecommendations. From compiled data of two measurements, microparticlesexhibited a unimodal distribution with all detectable events betweenabout 0.6 microns and 4 microns in volumetric mean diameter (VMD) andhaving a peak VMD at about 1.3 microns. By contrast, when BisEDT wasprepared by prior methods (Domenico et al., 1997 Antimicrob. AgentsChemother. 41(8):1697-1703) the majority of particles wereheterodisperse and of significantly larger size, precluding theircharacterization on the basis of VMD.

Example 2 Colony Biofilm Model of Chronic Wound Infection: Inhibition byBT Compounds

Because bacteria that exist in chronic wounds adopt a biofilm lifestyle,BTs were tested against biofilms for effects on bacterial cell survivalusing biofilms prepared essentially according to described methods(Anderl et al., 2003 Antimicrob Agents Chemother 47:1251-56; Walters etal., 2003 Antimicrob Agents Chemother 47:317; Wentland et al., 1996Biotchnol. Prog. 12:316; Zheng et al., 2002 Antimicrob Agents Chemother46:900).

Briefly, colony biofilms were grown on 10% tryptic soy agar for 24hours, and transferred to Mueller Hinton plates containing treatments.After treatment the biofilms were dispersed into peptone watercontaining 2% w/v glutathione (neutralizes the BT), and serially dilutedinto peptone water before being spotted onto plates for counting. Twobacteria isolated from chronic wounds were used separately in theproduction of colony biofilms for testing. These were Pseudomonasaeruginosa, a gram negative bacterial strain, and Methicillin ResistantStaphylococcus aureus (MRSA), which is gram positive.

Bacterial biofilm colonies were grown on top of micro porous membranesresting on an agar plate essentially as described (Anderl et al., 2003Antimicrob Agents Chemother 47:1251-56; Walters et al., 2003 AntimicrobAgents Chemother 47:317; Wentland et al., 1996 Biotchnol. Prog. 12:316;Zheng et al., 2002 Antimicrob Agents Chemother 46:900) The colonybiofilms exhibited many of the familiar features of other biofilmmodels, e.g., they consisted of cells densely aggregated in a highlyhydrated matrix. As also reported by others (Brown et al., J Surg Res56:562; Millward et al, 1989 Microbios 58:155; Sutch et al., 1995 JPharm Pharmacol 47:1094; Thrower et al., 1997 J Med Microbiol 46:425) itwas observed that bacteria in colony biofilms exhibited the sameprofoundly reduced anti-microbial susceptibility that has beenquantified in more sophisticated in vitro biofilm reactors. Colonybiofilms were readily and reproducibly generated in large numbers.According to non-limiting theory, this colony biofilm model shared someof the features of an infected wound: bacteria grew at an air interfacewith nutrients supplied from beneath the biofilm and minimal fluid flow.A variety of nutrients sources was used to cultivate colony biofilms,including blood agar, which is believed to mimic in vivo nutrientconditions.

Colony biofilms were prepared by inoculating 5 μl spots of planktonicbacterial liquid cultures onto a 25 mm diameter polycarbonate filtermembrane. The membranes were sterilized prior to inoculation, byexposure to ultraviolet light for 10 min per side. The inocula weregrown overnight in bacterial medium at 37° C. and diluted in freshmedium to an optical density of 0.1 at 600 nm prior to deposition on themembrane. The membranes were then placed on the agar plate containinggrowth medium. The plates were then covered and placed, inverted, in anincubator at 37° C. Every 24 h, the membrane and colony biofilm weretransferred, using sterile forceps, to a fresh plate. Colony biofilmswere typically used for experimentation after 48 hours of growth, atwhich time there were approximately 10⁹ bacteria per membrane. Thecolony biofilm method was successfully employed to culture a widevariety of single species and mixed species biofilms.

To measure susceptibility to antimicrobial agents (e.g., BT compoundsincluding combinations of BT compounds; antibiotics; and BTcompound-antibiotic combinations), colony biofilms were transferred toagar plates supplemented with the candidate antimicrobial treatmentagent(s). Where the duration of exposure to antimicrobial treatmentexceeded 24 hours, the colony biofilms were moved to fresh treatmentplates daily. At the end of the treatment period, the colony biofilmswere placed in tubes containing 10 ml of buffer and vortexed for 1-2 minto disperse the biofilm. In some cases, it was necessary to brieflyprocess the sample with a tissue homogenizer to break up cellaggregates. The resulting cell suspensions were then serially dilutedand plated to enumerate surviving bacteria, which were reported ascolony forming units (CFU) per unit area. Survival data were analyzedusing log₁₀ transformation.

For each type of bacterial biofilm colony cultures (Pseudomonasaeruginosa, PA; methicilin resistant Staphylococcus aureus, MRSA or SA)five antibiotics and thirteen BT compounds were tested. Antimicrobialagents tested against PA included the BTs referred to herein as BisEDTand Compounds 2B, 4, 5, 6, 8-2, 9, 10, 11 and 15 (see Table 1), and theantibiotics tobramycin, amikacin, imipenim, cefazolin, andciprofloxacin. Antimicrobial agents tested against SA included the BTsreferred to herein as BisEDT and Compounds 2B, 4, 5, 6, 8-2, 9, 10 and11 (see Table 1), and the antibiotics rifampicin, daptomycin,minocycline, ampicillin, and vancomycin. As described above under “briefdescriptions of the drawings”, antibiotics were tested at concentrationsof approximately 10-400 times the minimum inhibitory concentrations(MIC) according to established microbiological methodologies.

Seven BT compounds exhibited pronounced effects on PA bacterial survivalat the concentrations tested, and two BT compounds demonstratedpronounced effects on MRSA survival at the concentrations tested;representative results showing BT effects on bacterial survival arepresented in FIG. 1 for BisEDT and BT compound 2B (tested against PA)and in FIG. 2 for BT compounds 2B and 8-2 (tested against SA), in bothcases, relative to the effects of the indicated antibiotics. As alsoshown in FIGS. 1 and 2, inclusion of the indicated BT compounds incombination with the indicated antibiotics resulted in a synergisticeffect whereby the potency of reducing bacterial survival was enhancedrelative to the anti-bacterial effects of either the antibiotic alone orthe BT compound alone. In the PA survival assay, compound 15(Bis-EDT/2hydroxy, propane thiol (1:1/1)) at a concentration of 80 μg/mLexhibited an effect (not shown) that was comparable to the effectobtained using the combination of 1600 μg/mL AMK plus 80 μg/mL BisEDT(FIG. 1).

Example 3 Drip Flow Biofilm Model of Chronic Wound Infection: Inhibitionby BT Compounds

Drip flow biofilms represent an art accepted authentic model forforming, and testing the effect of candidate anti-bacterial compoundsagainst, bacterial biofilms. Drip flow biofilms are produced on coupons(substrates) placed in the channels of a drip flow reactor. Manydifferent types of materials can be used as the substrate for bacterialbiofilm formation, including frosted glass microscope slides. Nutritiveliquid media enters the drip flow bioreactor cell chamber by drippinginto the chamber near the top, and then flows the length of a coupondown a 10 degree slope.

Biofilms are grown in drip flow bioreactors and exposed to BT compoundsindividually or in combinations and/or to antibiotic compoundsindividually or in combinations with other antibacterial agents,including BT compounds, or to other conventional or candidate treatmentsfor chronic wounds. BT compounds are thus characterized for theireffects on bacterial biofilms in the drip-flow reactor. Biofilms in thedrip-flow reactor are prepared according to established methodologies(e.g., Stewart et al., 2001 J Appl Microbiol. 91:525; Xu et al., 1998Appl. Environ. Microbiol. 64:4035). This design involves cultivatingbiofilms on inclined polystyrene coupons in a covered chamber. Anexemplary culture medium contains 1 g/l glucose, 0.5 g/l NH₄NO₃, 0.25g/l KCl, 0.25 g/l KH₂PO₄, 0.25 g/l MgSO₄.7H₂O, supplemented with 5% v/vadult donor bovine serum (ph 6.8) that mimics serum protein-rich, ironlimited conditions that are similar to biofilm growth conditions invivo, such as in chronic wounds. This medium flows drop-wise (50 ml/h)over four coupons contained in four separate parallel chambers, each ofwhich measures 10 cm×1.9 cm by 1.9 cm deep. The chambered reactor isfabricated from polysulfone plastic. Each of the chambers is fitted withan individual removable plastic lid that can be tightly sealed. Thebiofilm reactor is contained in an incubator at 37° C., and bacterialcell culture medium is warmed by passing it through an aluminum heatsink kept in the incubator. This method reproduces the antibiotictolerant phenotype observed in certain biofilms, mimics the low fluidshear environment and proximity to an air interface characteristic of achronic wound while providing continual replenishment of nutrients, andis compatible with a number of analytical methods for characterizing andmonitoring the effects of introduced candidate antibacterial regimens.The drip-flow reactor has been successfully employed to culture a widevariety of pure and mixed-species biofilms. Biofilms are typically grownfor two to five days prior to application of antimicrobial agents.

To measure the effects of anti-biofilm agents on biofilms grown indrip-flow reactors, the fluid stream passing over the biofilm is amendedor supplemented with the desired treatment formulation (e.g., one ormore BT compounds and/or one or more antibiotics, or controls, and/orother candidate agents). Flow is continued for the specified treatmentperiod. The treated biofilm coupon is then briefly removed from thereactor and the biofilm is scraped into a beaker containing 10 ml ofbuffer. This sample is briefly processed (typically 30 s to 1 min) witha tissue homogenizer to disperse bacterial aggregates. The suspension isserially diluted and plated to enumerate surviving microorganismsaccording to standard microbiological methodologies.

Example 4 Wound Biofilm Inhibition of Keratinocyte Scratch Repair:Biofilm Suppression by BT Compounds

This Example describes a modification of established in vitrokeratinocyte scratch models of wound healing, to arrive at a modelhaving relevance to biofilm-associated wound pathology and woundhealing, and in particular to acute or chronic wounds or woundscontaining biofilms as described herein. According to the keratinocytescratch model of the effects of chronic wound biofilms, cultivation ofmammalian (e.g., human) keratinocytes and bacterial biofilm populationsproceeds in separate chambers that are in fluid contact with oneanother, to permit assessment of the effects of conditions thatinfluence the effects, of soluble components elaborated by biofilms, onkeratinocyte wound healing events.

Newborn human foreskin cells are cultured as monolayers in treatedplastic dishes, in which monolayers a controlled “wound” or scratch isformed by mechanical means (e.g., through physical disruption of themonolayer such as by scraping an essentially linear cell-free zonebetween regions of the monolayer with a suitable implement such as asterile scalpel, razor, cell scraper, forceps or other tool). In vitrokeratinocyte monolayer model systems are known to undergo cellularstructural and functional process in response to the wounding event, ina manner that simulates wound healing in vivo. According to the hereindisclosed embodiments, the influence of the presence of bacterialbiofilms on such processes, for instance, on the healing time of thescratch, is observed, and in these and related embodiments the effectsare also assessed of the presence of selected candidate antimicrobial(e.g., antibacterial and antibiofilm) treatments.

Wounded keratinocyte monolayers cultured in the presence of biofilms areexamined according to morphological, biochemical, molecular genetic,cell physiologic and other parameters to determine whether introductionof BT compounds alters (e.g., increases or decreases in a statisticallysignificant manner relative to appropriate controls) the damagingeffects of the biofilms. Wounds are first exposed to each BT compoundalone, and to contemplated combinations of BT compounds, in order totest the toxicity of each BT compound treatment prior to assessing theeffects of such treatments on biofilm influences toward the model woundhealing process.

In a representative embodiment, a three-day biofilm is cultured on amembrane (e.g., a membrane insert sold by Costar Corp. under thetrademark TRANSWELL®, or the like) that is maintained in a tissueculture well above, and in fluid communication with, a keratinocytemonolayer that is scratched to initiate the wound healing process.Biofilms cultured out of authentic acute or chronic wounds arecontemplated for use in these and related embodiments.

Thus, an in vitro system has been developed for evaluating solublebiofilm component effects on migration and proliferation of humankeratinocytes. The system separates the biofilm and keratinocytes usinga dialysis membrane. Keratinocytes are cultured from newborn foreskin aspreviously described (Fleckman et al., 1997 J. Invest. Dermatol. 109:36;Piepkorn et al., 1987 J. Invest. Dermatol. 88:215-219) and grown asconfluent monolayers on glass cover slips. The keratinocyte monolayerscan then be scratched to yield “wounds” with a uniform width, followedby monitoring cellular repair processes (e.g., Tao et al., 2007 PLoS ONE2:e697; Buth et al. 2007 Eur. J. Cell Biol. 86:747; Phan et al. 2000Ann. Acad. Med. Singapore 29:27). The artificial wounds are then placedin the bottom of a sterile double-sided chamber and the chamber isassembled using aseptic technique. Both sides of the chamber are filledwith keratinocyte growth medium (EpiLife) with or without antibioticsand/or bismuth-thiols. Uninoculated systems are used as controls.

The system is inoculated with wound-isolated bacteria and incubated instatic conditions for two hours to enable bacterial attachment tosurfaces in the upper chambers. Following the attachment period, liquidmedium flow is initiated in the upper chamber to remove unattachedcells. Flow of medium is then continued at a rate that minimizes thegrowth of planktonic cells within the upper chamber, by washout ofunattached cells. After incubation periods ranging from 6 to 48 hours,the systems (keratinocyte monolayers on coverslips and bacterial biofilmon membrane substrate) are disassembled and the cover slips removed andanalyzed. In related embodiments, mature biofilms are grown in the upperchamber prior to assembling the chamber. In other related embodiments,the separate co-culturing of biofilms and scratch-wounded keratinocytemonolayers is conducted in the absence and presence of one or more BTcompounds, optionally with the inclusion or exclusion of one or moreantibiotics, in order to determine effects of candidate agents such asBT compounds, or of potentially synergizing BT compound-plus-antibioticcombinations (e.g., a BT compound as provided herein such as a BT thatis provided in microparticulate form, and one or more of amikacin,ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin,clindamycin (or another lincoasamide antibiotic), daptomycin (sold underthe trademark CUBICIN®), doxycycline, gatifloxacin, gentamicin,imipenim, levofloxacin, linezolid (sold under the trademark ZYVOX®),minocycline, nafcilin, paromomycin, rifampin, sulphamethoxazole,tobramycin and vancomycin), on keratinocyte repair of the scratch wound,e.g., to identify an agent or combination of agents that alters (e.g.,increases or decreases in a statistically significant manner relative toappropriate controls) at least one indicator of scratch wound healing,such as the time elapsing for wound repair to take place or otherwound-repair indicia (e.g., Tao et al., 2007 PLoS ONE 2:e697; Buth etal. 2007 Eur. J Cell Biol. 86:747; Phan et al. 2000 Ann. Acad. Med.Singapore 29:27).

Example 5 Wound Biofilm Inhibition of Keratinocyte Scratch Repair

Isolated human keratinocytes were cultured on glass coverslips andscratch-wounded according to methodologies described above in Example 4.Wounded cultures were maintained under culture conditions alone or inthe presence of a co-cultured biofilm on a membrane support in fluidcommunication with the keratinocyte culture. The scratch closure timeinterval during which keratinocyte cell growth and/or migrationreestablishes the keratinocyte monolayer over the scratch zone was thendetermined. FIG. 3 illustrates the effect that the presence in fluidcommunication (but without direct contact) of biofilms had on thehealing time of scratched keratinocyte monolayers.

Accordingly there are contemplated in certain embodiments a method ofidentifying an agent for treating a chronic wound, comprising culturinga scratch-wounded cell (e.g., keratinocyte or fibroblast) monolayer inthe presence of a bacterial biofilm with and without a candidateanti-biofilm agent being present; and assessing an indicator of healingof the scratch-wounded cell monolayer in the absence and presence of thecandidate anti-biofilm agent, wherein an agent (e.g., a BT compound suchas a substantially monodisperse BT microparticle suspension as describedherein, alone or in synergizing combination with an antibiotic, such asone or more of amikacin, ampicillin, cefazolin, cefepime,chloramphenicol, ciprofloxacin, clindamycin, daptomycin (CUBICIN®),doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid(ZYVOX®), minocycline, nafcilin, paromomycin, rifampin,sulphamethoxazole, tobramycin and vancomycin) that promotes at least oneindicator of healing is identified as a suitable agent for treating anacute or chronic wound or a wound that contains a biofilm.

Example 6 Synergizing Bismuth-Thiol (BT)-Antibiotic Combinations

This example shows instances of demonstrated synergizing effects bycombinations of one or more bismuth-thiol compounds and one or moreantibiotics against a variety of bacterial species and bacterialstrains, including several antibiotic-resistant bacteria.

Materials & Methods. Susceptibility studies were performed by brothdilution in 96-well tissue culture plates (Nalge Nunc International,Denmark) in accordance with NCCLS protocols (National Committee forClinical Laboratory Standards. (1997). Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria that Grow Aerobically:Approved Standard M7-A2 and Informational Supplement M100-S10. NCCLS,Wayne, Pa., USA).

Briefly, overnight bacterial cultures were used to prepare 0.5 McFarlandstandard suspensions, which were further diluted 1:50 (˜2×10⁶ cfu/mL) incation-adjusted Mueller-Hinton broth medium (BBL, Cockeysville, Md.,USA). BTs (prepared as described above) and antibiotics were added atincremental concentrations, keeping the final volume constant at 0.2 mL.Cultures were incubated for 24 h at 37° C. and turbidity was assessed byabsorption at 630 nm using an ELISA plate reader (Biotek Instruments,Winooski, Vt., USA) according to the manufacturer's recommendations. TheMinimum Inhibitory Concentration (MIC) was expressed as the lowest drugconcentration inhibiting growth for 24 h. Viable bacterial counts(cfu/mL) were determined by standard plating on nutrient agar. TheMinimal Bactericidal Concentrations (MBC) was expressed as theconcentration of drug that reduced initial viability by 99.9% at 24 h ofincubation.

The checkerboard method was used to assess the activity of antimicrobialcombinations. The fractional inhibitory concentration index (FICI) andthe fractional bactericidal concentration index (FBCI) were calculated,according to Eliopoulos et al. (Eliopoulos and Moellering, (1996)Antimicrobial combinations. In Antibiotics in Laboratory Medicine(Lorian, V., Ed.), pp. 330-96, Williams and Wilkins, Baltimore, Md.,USA). Synergy was defined as an FICI or FBCI index of ≦0.5, nointeraction at >0.5-4 and antagonism at >4 (Odds, F C (2003) Synergy,antagonism, and what the chequerboard puts between them. Journal ofAntimicrobial Chemotherapy 52:1). Synergy was also definedconventionally as ≧4-fold decrease in antibiotic concentration.

Results are presented in Tables 2-17.

TABLE 2 S. aureus Nafcilin resistant NAF/BE NAF MIC MIC Strain (μg/ml)(μg/ml) Δ Synergy 60187-2 10.00 0.6 16.7 + 52446-3 175.00 40.0 4.4 +M1978 140.00 50.0 2.8 − W54793 130.00 33.3 3.9 − S24341 210.00 65.0 3.2− H7544 28.33 15.0 1.9 − H72751 145.00 43.3 3.3 − W71630 131.67 46.7 2.8− X22831 178.33 75.0 2.4 − X23660 123.33 43.3 2.8 − O36466 191.67 93.32.1 BE = 0.2 μg/ml BisEDT; Bacterial strains were obtained from theClinical Microbiology Laboratory at Winthrop-University Hospital,Mineola, NY. Nafcillin was obtained from Sigma (St. Louis, MO).

TABLE 3 S. aureus Nafcilin resistant GM/BE GM MIC MIC Strain (μg/ml)(μg/ml) Δ Synergy 60187-2 0.233 0.004 58.3 + 52446-3 10.667 1.500 7.1 +M1978 32.500 4.000 8.1 + W54793 0.250 0.080 3.1 − S24341 0.250 0.0584.3 + H7544 0.383 0.093 4.1 + H72751 0.200 0.072 2.8 − W71630 17.6673.800 4.6 + X22831 — 0.085 X23660 22.500 4.000 5.6 + O36466 0.267 0.0436.2 + BE = 0.2 μg/ml BisEDT; Bacterial strains were obtained from theClinical Microbiology Laboratory at Winthrop-University Hospital,Mineola, NY. Nafcillin was obtained from Sigma.

TABLE 4 S. aureus Rifampin/Neomycin/Paromomycin MIC MIC + BE (μg/ml)(μg/ml) Δ Synergy ATCC 25923 RIF 0.033 0.003 13.0 + NEO 0.500 0.200 2.5− PARO 1.080 0.188 5.7 + MRSA S2446-3 RIF 2.500 2.500 1.0 − NEO 13.4008.500 1.6 − PARO 335.000 183.300 1.8 − BE = 0.2 μg/ml BisEDT; StrainS2446-3 was obtained from the Clinical Microbiology Laboratory atWinthrop-University Hospital, Mineola, NY. Antibiotics were obtainedfrom Sigma.

TABLE 5 S. epidermidis - GM resistant strain ATCC 35984 strain S2400-1BisEDT MIC MBC MIC MBC (μg/ml) (μg/ml GM) (μg/ml GM) (μg/ml GM) (μg/mlGM) 0   53.3 384.0 85.3 426.7  0.005 20.0 96.0 96.0 512.0 0.01 37.3117.3 64.0 256.0 0.02 21.3 26.7 28.0 128.0 0.04 2.0 16.0 2.0 128.0 0.082.0 10.7 2.0 53.3 0.16 (MIC) 3.0 10.0 0.32 2.0 4.0 GM = gentamicin;Strain S2400-1 was obtained from the Clinical Microbiology Laboratory atWinthrop-University Hospital, Mineola, NY. Gentamicin was obtained fromthe Pharmacy Department at Winthrop; synergy in bold

TABLE 6 S. epidermidis - S2400-1 Biofilm Prevention BisEDT (μg/ml) ΔAntibiotic 0 0.05 0.1 (0.05 BE) Synergy cefazolin 28 10 1 2.8 −vancomycin 3.2 0.9 0.1 3.6 − gatifloxacin 1.6 0.1 0.1 16.0 ++ rifampicin0.03 0.04 0.04 0.7 − nafcillin 48 64 8 0.8 − clindamycin 1195 48 12 24.9++++ gentamicin 555 144 12 3.9 borderline minocycline 0.85 0.73 0.08 1.2− Data in μg/ml; Strain S2400-1 was obtained from the ClinicalMicrobiology Laboratory at Winthrop-University Hospital, Mineola, NY.Antibiotics were obtained from the Pharmacy Department at Winthrop.

TABLE 7 S. epidermidis - S2400-1 MIC BisEDT (μg/ml) Δ Antibiotic 0 0.050.1 (0.05 BE) Synergy cefazolin 32 8 1 4.00 + vancomycin 3.2 2.3 0.31.40 − gatifloxacin 1.7 0.8 0.3 2.13 − rifampicin 0.03 0.04 0.04 0.75 −nafcillin 171 192 68 0.89 − clindamycin 2048 768 24 2.67 − gentamicin2048 320 80 6.40 + minocycline 1.13 0.43 0.10 2.63 − Data in μg/ml;Strain S2400-1 was obtained from the Clinical Microbiology Laboratory atWinthrop-University Hospital, Mineola, NY. Antibiotics were obtainedfrom the Pharmacy Department at Winthrop.

TABLE 8 S. epidermidis - S2400-1 MBC BisEDT (μg/ml) Δ Antibiotic 0.0 0.1(0.1 BE) Synergy cefazolin 48 10 4.80 + vancomycin 5.4 1.4 3.86borderline gatifloxacin 2.8 1.4 2.00 − rifampicin 0.03 0.07 0.43 −nafcillin 256 128 2.00 − clindamycin 2048 768 2.67 − gentamicin 1536 2566.00 + minocycline 1.20 1.20 1.00 − Data in μg/ml; Strain S2400-1 wasobtained from the Clinical Microbiology Laboratory atWinthrop-University Hospital, Mineola, NY. Antibiotics were obtainedfrom the Pharmacy Department at Winthrop.

TABLE 9 S. epidermidis ATCC 35984 MIC BisEDT (μg/ml) Antibiotic 0.0 0.05Δ Synergy Nafcillin 16.00 5.00 3.2 − Clindamycin 2048.00 1024.00 2 −Gentamicin 213.33 16.00 13.3 ++ Minocycline 0.13 0.04 3.3 − Rifampicin0.021 0.014 1.5 − Data in μg/ml; Antibiotics were obtained from thePharmacy Department at Winthrop-University Hospital, Mineola, NY.

TABLE 10 E. coli - Ampicillin/Chloramphenicol resistant MIC MIC AB AB/BEMIC BE Strain (μg/ml) (μg/ml AB) Δ Synergy (μg/ml) MC4100/TN9 (CM) 22012.7 17.4 + 0.6 MC4100/P9 (AM) 285 49 5.8 + 0.5 MC4100 (AM) 141.7 354.0 + 0.6 AB = antibiotic; CM = chloramphenicol; AM = ampicillin; BE =BisEDT at 0.3 μg/ml; Strains were obtained from the laboratory of Dr. MJCasadaban, Department of Molecular Genetics and Cell Biology, TheUniversity of Chicago, Chicago, IL. Antibiotics were obtained from thePharmacy Department at Winthrop-University Hospital, Mineola, NY.

TABLE 11 E. coli - Tetracycline-resistant: Doxycycline + BisEDT DOX MICDOX/BE MIC BE MIC Strain (μg/ml) (μg/ml DOX) Δ Synergy (μg/ml) TET M16.50 4.50 4.0 + 0.85 TET D 20.50 0.03 820.0 ++++ 0.85 TET A 15.00 10.001.5 − 0.40 TET B 20.13 10.33 2.0 − 0.60 DOX = doxycycline; BE = BisEDTat 0.3 μg/ml; Strains were obtained from the laboratory of Dr. I Chopra,Department of Bacteriology, The University of Bristol, Bristol, UK.Antibiotics were obtained from the Pharmacy Department atWinthrop-University Hospital, Mineola, NY.

TABLE 12 P. aeruginosa - Tobramycin-resistant: BisEDT Synergy NN NN + BEBE MIC Strain (μg/ml) (μg/ml NN) Δ Synergy (μg/ml) Xen5 0.32 0.19 1.68 −0.9 Agr PA E 115 70 1.64 − 0.9 Agr PA I 200 73 2.74 − 1 Agr PA K 4.8 31.60 − 0.82 Agr PA O 130 20.5 6.34 + 0.98 Agr = aminoglycosideresistant; NN = tobramycin; PA = Pseudomonas aeruginosa; BE = BisEDT,0.3 μg/ml; Strains were obtained from the laboratory of Dr. K. Poole,Department of Microbiology and Immunology, Queens University, Ontario,CN. Tobramycin was obtained from the Pharmacy Department atWinthrop-University Hospital, Mineola, NY.

TABLE 13 B. cepacia Tobramycin + BE Synergy MIC NN NN + BE BE MIC Strain(μg/ml) (μg/ml NN) Δ Synergy (μg/ml) 13945 200 50 4 + 2.4 25416 125 1012.5 ++ 1.2 HI 2229 64 8 8 + 0.8 AU 0267 128 2 64 ++++ 0.8 AU 0259 1024256 4 + 1.6 HI 2255 64 8 8 + 1.6 AU 0273 512 32 16 ++ 1.6 HI 2253 64 164 + 1.6 HI 2147 512 8 64 ++++ 1.6 NN = Tobramycin; BE = BisEDT, 0.4μg/ml; Strains were obtained from the laboratory of Dr. J. J. LiPuma,Department of Pediatrics and Communicable Diseases, University ofMichigan, Ann Arbor, MI; also Veloira et al. 2003. Tobramycin wasobtained from the Pharmacy Department at Winthrop-University Hospital,Mineola, NY.

TABLE 14 B. cepacia Tobramycin + BE Synergy MBC NN NN + BE BE MIC Strain(μg/ml) (μg/ml NN) Δ Synergy (μg/ml) HI 2249 256 8 32 ++ 3.2 HI 2229 12832 4 + 6.4 AU 0267 256 32 8 + 6.4 AU 0259 1024 1024 1 − 12.8 HI 2255 12832 4 + 12.8 HI 2711 512 8 64 ++++ 6.4 AU 0284 1024 64 16 ++ 0.8 AU 0273512 32 16 ++ 1.6 HI 2253 128 64 2 − 3.2 HI 2147 512 128 4 + 6.4 NN =Tobramycin; BE = BisEDT, 0.4 μg/ml; Strains were obtained from thelaboratory of Dr. J. J. LiPuma, Department of Pediatrics andCommunicable Diseases, University of Michigan, Ann Arbor, MI; alsoVeloira et al. 2003. Tobramycin was obtained from the PharmacyDepartment at Winthrop-University Hospital, Mineola, NY.

TABLE 15 Tobramycin Resistant Strains MIC NN NN + BE Lipo-BE-NN Strain(μg/ml) (μg/ml NN) Δ Synergy (μg/ml NN) M13637 512 32 16 ++ 0.25 M13642R128 64 2 − 0.25 PA-48913 1024 256 4 + 0.25 PA-48912-2 64 8 8 + 0.25PA-10145 1 4 0.25 − 0.25 SA-29213 2 1 2 − 0.25 NN = Tobramycin; BE =BisEDT, 0.8 μg/ml; Lipo-BE-NN = liposomal BE-NN; Strains were obtainedfrom the laboratory of Dr. A. Omri, Department of Chemistry andBiochemistry, Laurentian University, Ontario, CN; (M strains are mucoidB. cepacia; PA = P. aeruginosa; SA = S. aureus). Tobramycin was obtainedfrom the Pharmacy Department at Winthrop-University Hospital, Mineola,NY.

TABLE 16 Tobramycin Resistant Strains MBC Lipo-BE- NN NN + BE NN Strain(μg/ml) (μg/ml NN) Δ Synergy (μg/ml NN) M13637 1024 64 16 ++ 8 M13642R256 128 2 − 16 PA-48913 4096 512 8 + 4 PA-48912-2 128 32 4 + 0.5PA-10145 1 8 0.125 − 4 SA-29213 2 1 2 − 0.25 NN = Tobramycin; BE =BisEDT, 0.8 μg/ml; Lipo-BE-NN = liposomal BE-NN; Strains were obtainedfrom the laboratory of Dr. A. Omri, Department of Chemistry andBiochemistry, Laurentian University, Ontario, CN; (M strains are mucoidB. cepacia; PA = P. aeruginosa; SA = S. aureus). Tobramycin was obtainedfrom the Pharmacy Department at Winthrop-University Hospital, Mineola,NY.

TABLE 17 BisEDT-Pyrithione Synergy S. aureus P. aeruginosa E. coli ATCCNaPYR ATCC 27853 ATCC 25922 25923 (ug/ml) (μg/ml BE) (μg/ml BE) (μg/mlBE) 0   0.25 0.1 0.25  0.025 0.1 0.125  0.05 0.025 0.063 0.1 0.1250.0125 0.063 0.2 0.125 0.0125 0.031 0.4 0.00625 0 0.8 0.125 0.00625 1.60.063 0.00625 (MIC) 3.2 0.063 0 6.4 0.063 12.8  0 BE = BisEDT; NaPYR =sodium pyrithione; Chemicals were obtained from Sigma-Aldrich; synergyin bold. Indicated bacterial strains were from American Type CultureCollection (ATCC, Manassas, VA).

Example 7 Comparative Bismuth-Thiol (BT) and Antibiotic Effects AgainstGram-Positive and Gram-Negative Bacteria Including Antibiotic-ResistantBacterial Strains

In this example the in vitro activities of BisEDT and comparator agentswere assessed against multiple clinical isolates of Gram-positiveand—negative bacteria that are responsible for skin and soft tissueinfections.

Materials and Methods. Test compounds and test concentration ranges wereas follows: BisEDT (Domenico et al., 1997; Domenico et al., Antimicrob.Agents Chemother. 45(5):1417-1421. and Example 1), 16-0.015 μg/mL;linezolid (ChemPacifica Inc., #35710), 64-0.06 μg/mL; Daptomycin (CubistPharmaceuticals # MCB2007), 32-0.03 μg/mL and 16-0.015 μg/mL; vancomycin(Sigma-Aldrich, St. Louis, Mo., # V2002), 64-0.06 μg/mL; ceftazidime,(Sigma # C3809), 64-0.06 μg/mL and 32-0.03 μg/mL; imipenem (UnitedStates Pharmacopeia, NJ, #1337809) 16-0.015 μg/mL and 8-0.008 μg/mL;ciprofloxacin (United States Pharmacopeia, # IOC265), 32-0.03 μg/mL and4-0.004 μg/mL; gentamicin (Sigma #G3632) 32-0.03 μg/mL and 16-0.015μg/mL. All test articles, except gentamicin, were dissolved in DMSO;gentamicin was dissolved in water. Stock solutions were prepared at40-fold the highest concentration in the test plate. The finalconcentration of DMSO in the test system was 2.5%.

Organisms. The test organisms were obtained from clinical laboratoriesas follows: CHP, Clarian Health Partners, Indianapolis, Ind.; UCLA,University of California Los Angeles Medical Center, Los Angeles,Calif.; GR Micro, London, UK; PHRI TB Center, Public Health ResearchInstitute Tuberculosis Center, New York, N.Y.; ATCC, American TypeCulture Collection, Manassas, Va.; Mt Sinai Hosp., Mount Sinai Hospital,New York, N.Y.; UCSF, University of California San Francisco GeneralHospital, San Francisco, Calif.; Bronson Hospital, Bronson MethodistHospital, Kalamazoo, Mich.; quality control isolates were from theAmerican Type Culture Collection (ATCC, Manassas, Va.). Organisms werestreaked for isolation on agar medium appropriate to each organism.Colonies were picked by swab from the isolation plates and put intosuspension in appropriate broth containing a cryoprotectant. Thesuspensions were aliquoted into cryogenic vials and maintained at −80°C. Abbreviations are: BisEDT, bismuth-1,2-ethanedithiol; LZD, linezolid;DAP, daptomycin; VA, vancomycin; CAZ, ceftazidime; IPM, imipenem; CIP,ciprofloxacin; GM, gentamicin; MSSA, methicillin-susceptibleStaphylococcus aureus; CLSI QC, Clinical and Laboratory StandardsInstitute quality control strain; MRSA, methicillin-resistantStaphylococcus aureus; CA-MRSA, community-acquired methicillin-resistantStaphylococcus aureus; MSSE, methicillin-susceptible Staphylococcusepidermidis; MRSE, methicillin-resistant Staphylococcus epidermidis;VSE, vancomycin-susceptible Enterococcus.

The isolates were streaked from the frozen vials onto appropriatemedium: Trypticase Soy Agar (Becton-Dickinson, Sparks, Md.) for mostorganisms or Trypticase Soy Agar plus 5% sheep blood (ClevelandScientific, Bath, Ohio) for streptococci. The plates were incubatedovernight at 35° C. Quality control organisms were included. The mediumemployed for the MIC assay was Mueller Hinton II Broth (MHB II-BectonDickinson, #212322) for most of the organisms. MHB II was supplementedwith 2% lysed horse blood (Cleveland Scientific Lot # H13913) toaccommodate the growth of Streptococcus pyogenes and Streptococcusagalactiae. The media were prepared at 102.5% normal weight to offsetthe dilution created by the addition of 5 μL drug solution to each wellof the microdilution panels. In addition, for tests with daptomycin, themedium was supplemented with an additional 25 mg/L Ca²⁺.

The MIC assay method followed the procedure described by the Clinicaland Laboratory Standards Institute (Clinical and Laboratory StandardsInstitute. Methods for Dilution Antimicrobial Susceptibility Tests forBacteria That Grow Aerobically; Approved Standard—Seventh Edition.Clinical and Laboratory Standards Institute document M7-A7 [ISBN1-56238-587-9]. Clinical and Laboratory Standards Institute, 940 WestValley Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 2006) and employedautomated liquid handlers to conduct serial dilutions and liquidtransfers. Automated liquid handlers included the MULTIDROP® 384(Labsystems, Helsinki, Finland), BIOMEK® 2000 and MULTIMEK™ 96 (BeckmanCoulter, Fullerton Calif.). The wells of Columns 2-12 of standard96-well microdilution plates (Falcon 3918) were filled with 150 μL ofDMSO or water for gentamicin on the MULTIDROP® 384. The drugs (300 μL)were dispensed into Column 1 of the appropriate row in these plates.These would become the mother plates from which the test plates(daughter plates) were prepared. The BIOMEK® 2000 completed serialtransfers through Column 11 in the mother plates. The wells of Column 12contained no drug and were the organism growth control wells in thedaughter plates. The daughter plates were loaded with 185 μL of theappropriate test media (described above) using the MULTIDROP® 384. Thedaughter plates were prepared on the MULTIMEK™ 96 instrument whichtransferred 5 μL of drug solution from each well of a mother plate toeach corresponding well of each daughter plate in a single step.

Standardized inoculum of each organism was prepared per CLSI methods(ISBN 1-56238-587-9, cited supra). Suspensions were prepared in MHB toequal the turbidity of a 0.5 McFarland standard. The suspensions werediluted 1:9 in broth appropriate to the organism. The inoculum for eachorganism was dispensed into sterile reservoirs divided by length(Beckman Coulter), and the BIOMEK® 2000 was used to inoculate theplates. Daughter plates were placed on the BIOMEK® 2000 work surfacereversed so that inoculation took place from low to high drugconcentration. The BIOMEK® 2000 delivered 10 μL of standardized inoculuminto each well. This yielded a final cell concentration in the daughterplates of approximately 5×10⁵ colony-forming-units/mL. Thus, the wellsof the daughter plates ultimately contained 185 μL of broth, 5 μL ofdrug solution, and 10 μL of bacterial inoculum. Plates were stacked 3high, covered with a lid on the top plate, placed in plastic bags, andincubated at 35° C. for approximately 18 hours for most of the isolates.The Streptococcus plates were read after 20 hours incubation. Themicroplates were viewed from the bottom using a plate viewer. For eachof the test media, an uninoculated solubility control plate was observedfor evidence of drug precipitation. The MIC was read and recorded as thelowest concentration of drug that inhibited visible growth of theorganism.

Results. All marketed drugs were soluble at all of the testconcentrations in both media. BisEDT exhibited a trace precipitate at 32μg/mL, but MIC readings were not affected as the inhibitoryconcentrations for all organisms tested were well below thatconcentration. On each assay day, an appropriate quality controlstrain(s) was included in the MIC assays. The MIC values derived forthese strains were compared to the published quality control ranges(Clinical and Laboratory Standards Institute. Performance Standards forAntimicrobial Susceptibility Testing; Eighteenth InformationalSupplement. CLSI document M100-S18 [ISBN 1-56238-653-0]. Clinical andLaboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,Pa. 19087-1898 USA, 2008) for each agent, as appropriate.

On each assay day, an appropriate quality control strain(s) was includedin the MIC assays. The MIC values derived for these strains werecompared to the published quality control ranges (Clinical andLaboratory Standards Institute. Performance Standards for AntimicrobialSusceptibility Testing; Eighteenth Informational Supplement. CLSIdocument M100-S18 [ISBN 1-56238-653-0]) for each agent, as appropriate.Of 141 values for quality control strains where quality control rangesare published, 140(99.3%) were within the specified ranges. The oneexception was imipenem versus S. aureus 29213 which yielded one value ona single run (≦0.008 μg/mL) that was one dilution below the published QCrange. All other quality control results on that run were within thespecified quality control ranges.

BisEDT demonstrated potent activity against both methicillin-susceptibleStaphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA),and community-acquired MRSA (CA-MRSA), inhibiting all strains tested at1 μg/mL or less with an MIC90 values of 0.5 μg/mL for all three organismgroups. BisEDT exhibited activity greater than that of linezolid andvancomycin and equivalent to that of daptomycin. Imipenem was morepotent than BisEDT against MSSA (MIC90=0.03 μg/mL). However, MRSA andCAMRSA were resistant to imipenem while BisEDT demonstrated activityequivalent to that shown for MSSA. BisEDT was highly-active againstmethicillin-susceptible and methicillin—resistant Staphylococcusepidermidis (MSSE and MRSE), with MIC90 values of 0.12 and 0.25 μg/mL,respectively. BisEDT was more active against MSSE than any of the otheragents tested except imipenem. BisEDT was the most active agent testedagainst MRSE.

BisEDT demonstrated activity equivalent to that of daptomycin,vancomycin, and imipenem against vancomycin-susceptible Enterococcusfaecalis (VSEfc) with an MIC90 value of 2 μg/mL. Significantly, BisEDTwas the most active agent tested against vancomycin-resistantEnterococcus faecalis (VREfc) with an MIC90 value of 1 μg/mL.

BisEDT was very active against vancomycin-susceptible Enterococcusfaecium (VSEfm) with an MIC90 value of 2 μg/mL; its activity wasequivalent to that or similar to that of daptomycin and one-dilutionhigher than that of vancomycin. BisEDT and linezolid were the mostactive agents tested against vancomycin-resistant Enterococcus faecium(VREfm), each demonstrating an MIC90 value of 2 μg/mL. The activity ofBisEDT against Streptococcus pyogenes (MIC90 value of 0.5 μg/mL) wasequivalent to that of vancomycin, greater than that of linezolid, andslightly less than that of daptomycin and ceftazidime. The compoundinhibited all strains tested at 0.5 μg/mL or less. In these studies, thespecies that was least sensitive to BisEDT was Streptococcus agalactiaewhere the observed MIC90 value was 16 μg/mL. BisEDT was less active thanall of the agents tested except gentamicin.

The activity of BisEDT and comparators against Gram-negative bacteriaincluded demonstrated BisEDT potency against Acinetobacter baumanii(MIC90 value of 2 μg/mL) making BisEDT the most active compound tested.Elevated MICs for a significant number of test isolates for thecomparator agents resulted in off-scale MIC90 values for these agents.BisEDT was a potent inhibitor of Escherichia coli, inhibiting allstrains at 2 μg/mL or less (MIC90=2 μg/mL). The compound was less activethan imipenem, but more active than ceftazidime, ciprofloxacin, andgentamicin. BisEDT also demonstrated activity against Klebsiellapneumoniae with an MIC90 value of 8 μg/mL which was equivalent to thatof imipenem. The relatively high MIC90 values exhibited by imipenem,ceftazidime, ciprofloxacin, and gentamicin indicated that this was ahighly antibiotic-resistant group of organisms. BisEDT was the mostactive compound tested against Pseudomonas aeruginosa with an MIC90value of 4 μg/mL. There was a high level of resistance to the comparatoragents for this group of test isolates.

In summary, BisEDT demonstrated broad-spectrum potency against multipleclinical isolates representing multiple species, including speciescommonly involved in acute and chronic skin and skin structureinfections in humans. The activity of BisEDT and key comparator agentswas evaluated against 723 clinical isolates of Gram-positive andGram-negative bacteria. The BT compound demonstrated broad spectrumactivity, and for a number of the test organisms in this study, BisEDTwas the most active compound tested in terms of anti-bacterial activity.BisEDT was most active against MSSA, MRSA, CA-MRSA, MSSE, MRSE, and S.pyogenes, where the MIC90 value was 0.5 μg/mL or less. Potent activitywas also demonstrated for VSEfc, VREfc, VSEfm, VREfm, A. baumanii, E.coli, and P. aeruginosa where the MIC90 value was in the range of 1-4μg/mL. MIC90 values observed were, for K. pneumoniae (MIC90=8 μg/mL),and for S. agalactiae (MIC90=16 μg/mL).

Example 8 Microparticulate BT-Antibiotic Enhancing and SynergizingActivities

This example shows that microparticulate bismuth thiols (BTs) promoteantibiotic activity through enhancing and/or synergizing interactions.

A major complicating factor in treating infections is the emergingresistance of bacteria to antibiotics. Methicillin resistance in S.epidermidis (MRSE) and S. aureus (MRSA) actually reflects multiple drugresistance, making these pathogens very difficult to eradicate. However,no staphylococci from hundreds of strains tested showed resistance toBTs. Furthermore, BTs at subinhibitory (subMIC) concentrations reducedresistance to several important antibiotics.

Staphylococcus aureus

A graphic demonstration of the antibiotic-resensitizing effects ofsubMIC bismuth ethanedithiol (BisEDT) against MRSA is provided (FIG. 4)showing enhanced antibiotic action of several classes of antibiotics,including gentamicin, cefazolin, cefepime, imipenim, sulphamethoxazole,and levofloxacin. Thus, BisEDT nonspecifically enhanced the activity ofmost antibiotics.

Broth dilution antimicrobial susceptibility studies were performedagainst 12 MRSA strains using several antibiotics combined with subMIClevels of BisEDT (Table 18). Both the biofilm-prevention concentration(BPC) and the minimum inhibitory concentration (MIC) were determined ina special biofilm culture medium (BHIG/X). The MIC and BPC forgentamicin and cefazolin were reduced by subMIC BisEDT (BisEDT MIC,0.2-0.4 μg/ml), but not below the breakpoint for sensitivity. subMICBisEDT enhanced the sensitivity of MRSA to gatifloxacin and cefepimeclose to the breakpoint for sensitivity. These strains were alreadysensitive to vancomycin, but were made considerably moreso in thepresence of subMIC BisEDT. Generally, the MIC and BPC were reduced 2- to5-fold with subMIC BisEDT.

TABLE 18 Antimicrobial Activity of BT-Antibiotic Combinations againstMRSA MIC Standards BisEDT (μg/mL) (μg/ml) Antibiotic 0 0.025 0.05 0.1 SR Gentamicin BPC 81 ± 41 63 ± 30 53 ± 31 33 ± 25 MIC 81 ± 40 60 ± 27 58± 30 48 ± 31 ≦4 ≧16 Cefazolin BPC 109 ± 86  76 ± 86  76 ± 105 34 ± 28MIC 93 ± 75 99 ± 76 90 ± 60 45 ± 32 ≦8 ≧32 Gatifloxacin BPC 3.6 ± 2.62.6 ± 0.9 2.4 ± 1.1 0.9 ± 0.8 MIC 3.6 ± 2.6 4.0 ± 2.8 4.0 ± 2.8 2.4 ±1.1 ≦2 ≧8 Vancomycin BPC 2.5 ± 1.7 1.5 ± 0.6 1.3 ± 0.5 0.7 ± 0.4 MIC 2.5± 1.7 2.5 ± 1.7 1.5 ± 0.6 1.3 ± 0.5 ≦4 ≧32 Cefepime BPC 24 ± 37 27 ± 2818 ± 16 5.0 ± 7.3 MIC 45 ± 32 32 ± 28 37 ± 24 9.3 ± 6.1 ≦8 ≧32

-   -   12 MRSA clinical isolates were grown in BHIG/X and exposed to        serial dilutions of antibiotics in the presence of 0-0.1 μg/ml        BisEDT. The MIC and BPC, calculated in μg/ml, are the        means±standard deviations from at least three trials. The right        hand column lists the Standard MIC for antibiotic        sensitivity (S) and resistance (R)

A broth dilution study of cefepime-resistant MRSA isolates is shown inTable 19. BisEDT at 0.1 μg/ml significantly enhanced the inhibitoryactivity of cefepime in 11 of 12 isolates. In this particular study, thedata indicated synergy between BisEDT and cefepime (FIC<0.5), with manyof the isolates at the breakpoint for sensitivity.

TABLE 19 Cefepime-resistant MRSA Sensitized by BisEDT MIC for Cefepime(ug/mL) in subMIC BisEDT BE BE BE 0 μg/mL 0.05 μg/mL 0.1 μg/mL MRSA MICMIC MIC Strain #  4 256 256 16  6 256 256 32  7 128 256 32 10 128 32 1618 256 128 8 24 256 64 8 28 256 128 8 35 256 256 8 37 128 128 8 41 128256 8 46 256 256 256 47 32 8 8

-   -   Twelve cefepime-resistant MRSA were tested in BHIG/X medium in        polystyrene plates for sensitivity to cefepime combined with        subMIC BisEDT at 37° C. for 48 h.

Results for combination studies with nafcillin or gentamicin are shownin Table 20. Combined with nafcillin, BisEDT (0.2 μg/ml) reduced theMIC90 for nafcillin by over 4-fold against MRSA (FIC, 0.74). Combinedwith gentamicin, BisEDT reduced the MIC90 for gentamicin over 10-foldagainst MRSA (FIC, 0.6). BTs reversed the resistance of all fourgentamicin-resistant isolates tested to clinically relevantconcentrations [Domenico et al., 2002]. The MICs for these antimicrobialagents was reduced substantially, especially for gentamicin. The brothused in these studies was Trypticase Soy Broth (TSB) with 2% glucose,which showed results similar to that seen in Mueller-Hinton II brothfortified with 1% sheep's blood.

TABLE 20 MRSA: Nafcillin or Gentamicin + BisEDT Synergy NAF NAF + BE GMGM + BE Strain MIC MIC Δ MIC MIC Δ 60187-2 10.00 0.60 16.67 0.23 0.0058.33 52446-3 175.00 40.00 4.38 10.67 1.50 7.11 M1978 140.00 50.00 2.8032.50 4.00 8.13 W54793 130.00 33.33 3.90 0.25 0.08 3.13 S24341 210.0065.00 3.23 0.25 0.06 4.29 H7544 28.33 15.00 1.89 0.38 0.09 4.11 H72751145.00 43.33 3.35 0.20 0.07 2.79 W71630 131.67 46.67 2.82 17.67 3.804.65 X22831 178.33 75.00 2.38 X23660 123.33 43.33 2.85 22.50 4.00 5.63O36466 191.67 93.33 2.05 0.27 0.04 6.15 AVG Δ 4.21 AVG Δ 10.43 NAF or GMin μg/ml; BE at 0.2 μg/ml

Staphylococcus epidermidis

The activities of most classes of antibiotic were promoted in thepresence of BisEDT. With regard to the BPC, clindamycin and gatifloxacinshowed significantly more antibiofilm activity against S. epidermidiswhen combined with BisEDT (FIG. 5). Stated in different terms, the BPCfor clindamycin, gatifloxacin and gentamicin were reduced 50-fold,10-fold and 4-fold, respectively, in the presence of subMIC BisEDT.

Only modest decreases in the biofilm prevention concentration (BPC) werenoted for minocycline, vancomycin, and cefazolin, while rifampicin andnafcillin remained unaffected at 0.05 μg/ml BisEDT. At 0.1 μg/ml BisEDTno biofilm was detected, regardless of antibiotic employed, signifyingthat no antagonism occurred. This BisEDT concentration was close to theMIC for S. epidermidis [Domenico et al., 2003] (See FIG. 5).

With regard to growth inhibition, seven of eight antibiotics tested weresignificantly enhanced in the presence of 0.1 μg/ml (0.5 μM) BisEDTagainst S. epidermidis (FIG. 6). The MIC change was most pronounced forclindamycin and gentamicin, followed by vancomycin, cefazolin,minocycline, gatifloxacin and nafcillin, with rifampicin unaffected. Ofthe antibiotics this strain was resistant to (NC, CZ, GM, CM), onlycefazolin resistance was reversed to clinically relevant levels byBisEDT.

Minimum bactericidal concentration (MBC) for most antibiotics testedagainst S. epidermidis decreased slightly with subMIC BisEDT. Gentamicinshowed the greatest reduction in MBC (4- to 16-fold), followed bycefazolin (4- to 5-fold), vancomycin and nafcillin (3- to 4-fold),minocycline and gatifloxacin (2- to 3-fold), while clindamycin andrifampicin MBC remained largely unaffected. Clindamycin is abacteriostatic agent, which explains its lack of bactericidal activity.Cefazolin resistance was reversed with respect to the MBC [Domenico etal., 2003]. These effects were additive.

The potentiation of antimicrobial agents was also demonstrated in vivoin a graft infection rat model (Table 21). BisEDT levels as low as 0.1μg/ml were able to promote the prevention of resistant S. epidermidisbiofilm for 7 days.

As summarized in Table 21, implants impregnated with 0.1 μg/ml BisEDT,10 μg/ml RIP and 10 μg/ml rifampin, alone or combined were implanteds.c. into rats. Physiological solution (1 ml) containing the MS and MRstrains at 2×10⁷ cfu/ml was inoculated onto the graft surface using atuberculin syringe. All grafts were explanted at 7 days followingimplantation and sonicated for 5 minutes in sterile saline solution toremove the adherent bacteria. Quantitation of viable bacteria wasobtained by culturing dilutions on blood agar plates. The limit ofdetection was approximately 10 cfu/cm².

TABLE 21 RIP, BTs, and rifampin against S. epidermidis in a graftinfection model Quantitative graft culture Group^(a) Graft-bondeddrug^(b) (cfu/cm²) No MSSE — <10 Untreated MSSE — 5.0 × 10⁷ ± 7.7 × 10⁶MS1^(c) RIP 4.3 × 10² ± 1.2 × 10² MS2^(c) BTs 5.8 × 10² ± 0.9 × 10²MS3^(c) Rifampin 5.9 × 10³ ± 1.8 × 10³ MS^(cd) RIP plus BTs <10 MS5^(cd)RIP plus rifampin 2.0 × 10¹ ± 0.6 × 10¹ MS6^(cd) BTs plus rifampin 1.9 ×10¹ ± 0.4 × 10¹ No MRSE — <10 Untreated MRSE — 7.8 × 10⁷ ± 2.0 × 10⁷MR1^(c) RIP 6.7 × 10² ± 2.1 × 10² MR2^(c) BTs 6.2 × 10² ± 2.3 × 10²MR3^(c) Rifampin 7.6 × 10⁴ ± 2.1 × 10⁴ MR4^(ce) RIP plus BTs <10 MR5^(c)RIP plus rifampin 4.3 × 10¹ ± 1.1 × 10¹ MR6^(c) BTs plus rifampin 3.0 ×10¹ ± 1.1 × 10¹ ^(a)Each group had 15 animals; MS,methicillin-susceptible S. epidermidis; MR, methicillin-resistant S.epidermidis ^(b)Dacron graft segments impregnated with 0.1 mg/l of BTs,10 mg/l of RIP, 10 mg/l of rifampin ^(c)Statistically significant whencompared with control groups MS and MR ^(d)Statistically significantwhen compared with MS3 group ^(e)Statistically significant when comparedwith MR1, MR2, and MR3 groups

Gram-Negative Bacteria.

Tobramycin activity against resistant Pseudomonas aeruginosa wasenhanced several-fold with subMIC BisEDT (Table 22). In these trials,the MIC was defined more precisely as the IC₂₄.

TABLE 22 Tobramycin-resistant P. aeruginosa: BisEDT Effect NN MIC BE MICNN + BE MIC Strain (μg/ml) (μg/ml) (μg/ml) Δ PA Xen5 0.3 0.9 0.2 1.7 AgrPA E 115.0 0.9 70.0 1.6 Agr PA I 200.0 1.0 73.0 2.7 Agr PA K 4.8 0.863.0 1.6 Agr PA O 130.0 0.98 20.5 6.3

-   -   Resistant strains of P. aeruginosa were cultured in        Mueller-Hinton II broth at 37° C. in the presence of tobramycin        (NN) and BisEDT (BE; 0.33 μg/ml). The MIC was determined as the        antibiotic concentration that inhibited growth for 24±1 h.

Against tobramycin-resistant Burkholderia cepacia, 0.4 μg/ml BisEDTrendered seven of 10 isolates tobramycin sensitive (mean FIC; 0.48), andreduced the MIC₉₀ by 10-fold (Table 23). Both the MIC and MBC oftobramycin were reduced significantly to achievable levels against 50clinical Burkholderia cepacia isolates with subMIC BisEDT [Veloira etal., 2003]. BisEDT and tobramycin in liposomal form have proven highlysynergistic against P. aeruginosa. (Halwani et al., 2008; Halwani etal., 2009).

TABLE 23 Tobramycin and BisEDT versus B. cepacia MIC for TobramycinBisEDT Tobramycin FIC Strain (μg/ml) (μg/ml) (BisEDT at 0.4 μg/ml) IndexB. multivorans HI 2249 256 0.4 a a HI 2229 64 0.8 8 0.63 AU 0267 128 0.82 0.52 AU 0259 1024 1.6 256 0.50 HI 2255 64 1.6 8 0.38 B. cenocepacia HI2711 256 0.4 a a AU 0284 512 0.4 a a AU 0273 512 1.6 32 0.31 HI2253 641.6 16 0.50 HI 2147 512 1.6 8 0.27 a The three strains inhibited byBisEDT at 0.4 μg/ml were excluded from further study. FIC Index ≦0.5indicates synergy: FICI >0.5 and <1.0 indicates enhancement.

Chloramphenicol and ampicillin resistant Escherichia coli were madesensitive to these drugs by the addition of subMIC BisEDT (Table 24).

TABLE 24 Chloramphenicol/Ampicillin Resistant E. coli: BisEDT EffectDrug + BE Drug MIC BE MIC MIC Strain Drug (μg/ml) (μg/ml) (μg/ml) ΔMC4100/TN9 CM 220.0 0.6 12.7 17.4 MC4100/P9 AMP 285.0 0.5 49.0 5.8MC4100 AMP 141.7 0.6 35.0 4.0

-   -   Resistant strains of E. coli were cultured in Mueller-Hinton II        broth at 37° C. in the presence of chloramphenicol (CM) or        ampicillin (AMP) and BisEDT alone or in combination (BE; 0.33        μg/ml). The MIC was determined as the antibiotic concentration        that inhibited growth for 24±1 h.

Tetracycline resistant Escherichia coli were made sensitive todoxycycline by the addition of subMIC BisEDT (Table 25). The combinationexhibited synergy against the TET M and TET D strains (FIC≦0.5), withadditive effects against the TET A and TET B strains.

TABLE 25 Tetracycline Resistant E. Coli: BisEDT Effect DOX MIC BE MICDOX + BE MIC Strain (μg/ml) (μg/ml) (μg/ml) Δ TET M 16.5 ± 1.3 0.85  4.5± 2.7 4.0 TET D 20.5 ± 1.1 0.85 0.03 ± 0.0 820.0 TET A 15.0 ± 1.8 0.4010.0 ± 1.0 1.5 TET B 20.1 ± 2.4 0.60 10.3 ± 3.2 2.0

-   -   Resistant strains of E. coli were cultured in Mueller-Hinton II        broth at 37° C. in the presence of doxycycline (DOX) and BisEDT        alone or in combination (BE; 0.33 μg/ml). The MIC was determined        as the antibiotic concentration that inhibited growth for 24±1        h.

REFERENCES

Domenico P, R O'Leary, B A Cunha. 1992. Differential effect of bismuthand salicylate compounds on antibiotic sensitivity of Pseudomonasaeruginosa. Eur J Clin Microbiol Infec Dis 11:170-175; Domenico P, DParikh, B A Cunha. 1994. Bismuth modulation of antibiotic activityagainst gastrointestinal bacterial pathogens. Med Microbiol Lett3:114-119; Domenico P, Kazzaz J A, Davis J M, Niederman M S. 2002.Subinhibitory bismuth ethanedithiol (BisEDT) sensitizes resistantStaphylococcus aureus to nafcillin or gentamicin. Annual Meeting, ASM,Salt Lake City, Utah; Domenico P, Kazzaz J A, Davis J M. 2003. Combatingantibiotic resistance with bismuth-thiols. Research Advances inAntimicrob Agents Chemother 3:79-85; Domenico P, E Gurzenda, AGiacometti, O Cirioni, R Ghiselli, F Orlando, M Korem, V Saba, GScalise, N Balaban. 2004. BisEDT and RIP act in synergy to prevent graftinfections by resistant staphylococci. Peptides 25:2047-2053; Halwani M,Blomme S, Suntres Z E, Alipour M, Azghani A O, Kumar A, Omri A. 2008.Liposomal bismuth-ethanedithiol formulation enhances antimicrobialactivity of tobramycin. Intl J Pharmaceut 358:278-84; Halwani M, HebertS, Suntres Z E, Lafrenie R M, Azghani A O, Omri A. 2009. Bismuth-thiolincorporation enhances biological activities of liposomal tobramycinagainst bacterial biofilm and quorum sensing molecules production byPseudomonas aeruginosa. Int J Pharmaceut 373:141-6; Veloira W G,Gurzenda E M, Domenico P, Davis J M, Kazzaz J A. 2003. Synergy oftobramycin and bismuth thiols against Burkholderia cepacia. J AntimicrobChemother 52:915-919.

Example 9 Microparticulate BT-Antibiotic Enhancing and SynergizingActivities

This example shows that the microparticulate bismuth thiol BisEDTpromotes antibiotic activity through enhancing and/or synergizinginteractions with specific antibiotics against specific microbial targetorganisms. Single-point data for each indicated combination in Table 26were generated essentially according to the methods used in Example 8.

TABLE 26 FICI Values for single-point BisEDT-antibiotic combinationsAnti- SA MRSA E Fc SP PRSP EC EC KP PA Bcep Bmult Abau Msmeg biotic 100773 3121 1195 5348 102 2232 1231 1380 1756 5665 2594 817 Oxacillin 1.282.28 0.92 1.03 Piperacillin 0.57 1.28 1.11 1.11 0.87 1.29 2.23 0.67 1.121.12 1.12 Cefuroxime 1.11 4.23 1.11 1.03 Cefotaxime 1.11 2.23 0.73 1.111.11 1.37 1.29 0.61 0.64 1.29 1.11 1.29 Cefepime 0.87 0.96 1.11 0.621.34 0.96 0.71 Imipenem 0.67 1.48 0.73 0.92 0.43 1.11 1.29 1.23 1.120.73 1.23 0.81 Aztreonam 0.74 1.29 0.73 0.55 0.67 0.96 0.87 Streptomycin0.95 0.61 0.66 1.29 1.04 1.98 1.37 1.12 2.62 1.13 Tobramycin 0.73 0.780.47 0.57 0.96 0.87 1.29 0.91 0.67 1.12 Tetracycline 0.89 1.23 0.92 1.230.34 0.62 0.79 1.29 1.29 1.96 1.12 1.12 Minocycline 1.09 1.23 1.11 0.461.37 1.04 1.29 0.99 2.23 1.12 1.29 Ciprofloxacin 1.14 1.29 1.29 2.752.23 2.29 1.04 Levofloxacin 1.23 1.11 1.08 0.95 0.70 Erythromycin 1.280.67 0.92 0.78 1.03 Linezolid 1.23 1.23 1.23 1.01 1.11 Phosphomycin 0.611.23 1.45 1.96 1.02 1.86 1.29 1.23 1.12 Capreomycin 0.75 Isoniazid 0.88

SA, Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcusaureus; E Fc, Enterococcus faecalis; SP, Streptococcus pneumoniae; PRSP,penicillin-resistant Streptococcus pneumoniae; EC, Escherichia coli; KP,Klebsiella pneumoniae; PA, Pseudomonas aeruginosa; Bcep, Burkholderiacepacia; Bmult, Bukholderia multivorans; Abau, Acinetobacter baumanii;Msmeg, Mycobacterium smegmatis.

Example 10 Microparticulate BT-Antibiotic Enhancing and SynergizingActivities

The effects of combinations of microparticulate Bis-EDT and four Bis-EDTanalogs prepared as described above, and other agents againstrepresentative strains of several Gram-negative pathogenic bacteria weretested. A modification of a common laboratory method was used todetermine synergism (FICI≦0.5), enhancement (0.5<FICI≦1.0), antagonism(FICI>4.0) and indifference (1.0<FICI≦4.0) used fractional inhibitoryconcentrations (FICs) and FIC indices (FICI) (Eliopoulos G and RMoellering. 1991. Antimicrobial combinations. In Antibiotics inLaboratory Medicine, Third Edition, edited by V Lorian. Williams andWilkins, Baltimore, Md., pp. 432-492; Odds, 2003 J. Antimicrob.Chemother. 52(1):1). The checkerboard technique was used to determineFIC indices and was employed in this study.

TABLE 27 Test Components FIC Highest Stock Conc. Range ConcentrationTested in FIC Test Cpd Lot No. Solvent (μg/mL) (μg/mL) Bis-EDT MB-1B-3DMSO 320 0.12-8 Bis-EDT (analog) MB-2B DMSO 320 0.12-8 Bis-EDT (analog)MB-8-2 DMSO 320 0.12-8 Bis-EDT (analog) MB-11 DMSO 320 0.12-8 Bis-EDT(analog) MB-15 DMSO 320 0.12-8 Aztreonam 095K1324 10% 2,560  0.06-64(Sigma) DMSO Cefepime HCl GOD116 dH₂O 2,560  0.06-64 (USP) Cefotaxime084K0674 dH₂O 640 0.015-16 (Sigma) Piperacillin 014K1362 dH₂O 2,560 0.06-64 (Sigma)

Stock solutions of all test articles were prepared at 40× the finaltarget concentration in the appropriate solvent. All test articles werein solution under these conditions. The final drug concentrations in theFIC assay plates were set to bracket the MIC value of each agent foreach test organism, unless the strain was totally resistant to the testagent. The concentration ranges tested are displayed in Table 27. Thetest organisms were originally received from clinical sources, or fromthe American Type Culture Collection. Upon receipt, the isolates werestreaked onto Tryptic Soy Agar II (TSA). Colonies were harvested fromthese plates and a cell suspension was prepared in an appropriate brothgrowth medium containing cryoprotectant. Aliquots were then frozen at−80° C. The frozen seeds of the organisms to be tested in a given assaywere thawed, streaked for isolation onto TSA plates, and incubated at35° C. All organisms were tested in Mueller Hinton II Broth (BectonDickinson, Lot No. 9044411). The broth was prepared at 1.05× normalweight/volume to offset the 5% volume of the drugs in the final testplates.

Minimal Inhibitory Concentration (MIC) values were previously determinedusing the broth microdilution method for aerobic bacteria (Clinical andLaboratory Standards Institute (CLSI). Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;Approved Standard—Eighth Edition. CLSI document M07-A8 [ISBN1-56238-689-1]. Clinical and Laboratory Standards Institute, 940 WestValley Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 2009.).

FIC values were determined using a broth microdilution method previouslydescribed (Sweeney et al., 2003 Antimicrob. Agents Chemother.47(6):1902-1906). To prepare the test plates, automated liquid handlers(MULTIDROP® 384, Labsystems, Helsinki, Finland; BIOMEK® 2000 andMULTIMEK™ 96, Beckman Coulter, Fullerton Calif.) were used to conductserial dilutions and liquid transfers.

The appropriate wells of standard 96-well microdilution plates (FALCON™3918) were filled with 150 μL of the appropriate solvent in columns 2-12using the MULTIDROP® 384. Three hundred microliters of each secondarytest drug was added to each well in Column 1 of the plates. These plateswere used to prepare the drug “mother plates” which provided the serialdrug dilutions for the drug combination plates. The BIOMEK® 2000 wasused to transfer 150 μL of each secondary drug solution (40×) from thewells in Column 1 of the mother plate and to make eleven 2-fold serialdilutions. Mother plates of Bis-EDT (and analogs) were serial dilutedtop to bottom by hand, using a multichannel pipette. Two mother plates,one for each secondary drug and one for Bis-EDT (or analogs), werecombined to form a “checkerboard” pattern by transfer of equal volumes(using a multi-channel pipette) to the drug combination plate. Row H andColumn 12 each contained serial dilutions of one of the agents alone fordetermination of the MIC.

The “daughter plates” were loaded with 180 μL of test medium using theMULTIDROP® 384. Then, the MULTIMEK™ 96 was used to transfer 10 μL ofdrug solution from each well of the drug combination mother plate toeach corresponding well of the daughter plate in a single step. Finally,the daughter plates were inoculated with test organism. Standardizedinoculum of each organism was prepared per published guidelines (CLSI,2009). For all isolates, the inoculum for each organism was dispensedinto sterile reservoirs divided by length (Beckman Coulter), and theBIOMEK® 2000 was used to inoculate the plates. The instrument delivered10 μL of standardized inoculum into each well to yield a final cellconcentration in the daughter plates of approximately 5×10⁵colony-forming-units/mL.

The test format resulted in the creation of an 8×12 checkerboard whereeach compound was tested alone (Column 12 and Row H) and in combinationat varying ratios of drug concentration. All organism plates werestacked three high, covered with a lid on the top plate, placed inplastic bags, and incubated at 35° C. for approximately 20 hours.Following incubation, the microplates were removed from the incubatorsand viewed from the bottom using a plate viewer sold under the trademarkSCIENCEWARE® (Bet-Art Products Corp., South Wayne, N.J.). Preparedreading sheets were marked for the MIC of drug 1 (row H), the MIC ofdrug 2 (column 12) and the wells of the growth-no growth interface.

An EXCEL® program (Microsoft, Redmond, Wash.) was used to determine theFIC according to the formula: (MIC of Compound 1 in combination/MIC ofCompound 1 alone)+(MIC of Compound 2 in combination/MIC of Compound 2alone). The FICI for the checkerboard was calculated from the individualFICs by the formula: (FIC₁+FIC₂+FIC_(n))/n, where n=number of individualwells per plate for which FICs were calculated. In instances where anagent alone yielded an off-scale MIC result, the next highestconcentration was used as the MIC value in the FIC calculation.

Microparticulate Bis-EDT, the four microparticulate BT analogs, and allof the other agents (and combinations of agents) were soluble at allfinal test concentrations. The MIC and FICI values that were determinedare presented in the Tables below.

TABLE 28 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-1B-3 and Piperacillin Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-1B-3 1 Piperacillin >64 0.83 P. aeruginosa1384 1 8 0.96 P. aeruginosa 1474 1 8 0.71 P. aeruginosa 1479 0.5 8 1.12P. aeruginosa 2566 0.5 32 1.37 P. aeruginosa 2568 1 8 0.71 P. aeruginosa103 1 8 0.79 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 29 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-1B-3 and Aztreonam Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-1B-3 1 Aztreonam 32 1.04 P. aeruginosa 13841 8 0.71 P. aeruginosa 1474 1 8 0.71 P. aeruginosa 1479 0.5 8 0.87 P.aeruginosa 2566 0.5 16 1.37 P. aeruginosa 2568 1 8 0.71 P. aeruginosa103 1 4 1.29 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 30 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-15 and Piperacillin Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-15 1 Piperacillin >64 1.29 P. aeruginosa1384 1 16 0.71 P. aeruginosa 1474 1 8 1.12 P. aeruginosa 1479 1 8 1.29P. aeruginosa 2566 1 32 1.04 P. aeruginosa 2568 1 8 1.12 P. aeruginosa103 2 8 0.73 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 31 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-15 and Aztreonam Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-15 2 Aztreonam 32 1.11 P. aeruginosa 1384 18 0.79 P. aeruginosa 1474 1 8 0.71 P. aeruginosa 1479 2 8 0.67 P.aeruginosa 2566 0.5 16 1.12 P. aeruginosa 2568 1 8 0.79 P. aeruginosa103 2 4 1.23 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 32 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-8-2 and Piperacillin Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-8-2 2 Piperacillin >64 1.23 P. aeruginosa1384 2 16 0.73 P. aeruginosa 1474 2 8 1.23 P. aeruginosa 1479 2 8 1.23P. aeruginosa 2566 2 32 1.23 P. aeruginosa 2568 2 8 0.98 P. aeruginosa103 4 8 1.19 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 33 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-8-2 and Aztreonam Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-8-2 2 Aztreonam 32 1.11 P. aeruginosa 1384 28 1.11 P. aeruginosa 1474 2 8 0.73 P. aeruginosa 1479 2 8 0.98 P.aeruginosa 2566 2 16 1.23 P. aeruginosa 2568 2 8 0.98 P. aeruginosa 1034 8 1.19 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 34 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-11 and Piperacillin Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-11 1 Piperacillin >64 1.12 P. aeruginosa1384 1 16 0.71 P. aeruginosa 1474 1 8 1.12 P. aeruginosa 1479 1 8 1.29P. aeruginosa 2566 0.5 32 1.12 P. aeruginosa 2568 1 8 1.12 P. aeruginosa103 2 8 1.11 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 35 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-11 and Aztreonam Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-11 2 Aztreonam 32 0.92 P. aeruginosa 1384 18 0.96 P. aeruginosa 1474 1 8 0.71 P. aeruginosa 1479 1 8 0.79 P.aeruginosa 2566 0.5 16 1.12 P. aeruginosa 2568 1 8 0.96 P. aeruginosa103 2 8 1.11 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 36 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-2B and Piperacillin Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-2B 2 Piperacillin >64 1.02 P. aeruginosa1384 8 16 0.79 P. aeruginosa 1474 8 8 0.91 P. aeruginosa 1479 8 8 1.08P. aeruginosa 2566 8 32 1.04 P. aeruginosa 2568 8 8 0.97 P. aeruginosa103 8 8 1.16 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 37 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-2B and Aztreonam Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-2B 8 Aztreonam 64 0.89 P. aeruginosa 1384 88 0.91 P. aeruginosa 1474 8 8 0.54 P. aeruginosa 1479 8 8 0.87 P.aeruginosa 2566 8 16 0.91 P. aeruginosa 2568 8 8 0.87 P. aeruginosa 1038 8 1.08 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 38 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-1B-3 and Cefotaxime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² K. pneumoniae MB-1B-3 2 Cefotaxime 0.06 1.23 1346 K. pneumoniae 10.06 2.29 1355 K. pneumoniae 1 16 1.29 2238 K. pneumoniae 2 0.12 1.232541 K. pneumoniae 1 0.25 1.12 2546 K. pneumoniae 1 0.12 0.79 2549 P.aeruginosa 1 16 0.96 103 ¹MIC, Minimum Inhibitory Concentration ²FICI,Fractional Inhibitory Concentration Index

TABLE 39 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-1B-3 and Cefepime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-1B-3 1 Cefepime 32 1.29 P. aeruginosa 1384 12 0.79 P. aeruginosa 1474 1 2 0.79 P. aeruginosa 1479 1 4 1.12 P.aeruginosa 2566 0.5 8 1.37 P. aeruginosa 2568 1 2 0.79 P. aeruginosa 1031 2 0.71 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 40 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-15 and Cefotaxime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² K. pneumoniae 1346 MB-15 2 Cefotaxime 0.06 1.23 K. pneumoniae 13551 0.12 2.37 K. pneumoniae 2238 2 16 1.23 K. pneumoniae 2541 2 0.12 1.23K. pneumoniae 2546 2 0.25 0.97 K. pneumoniae 2549 2 0.06 1.23 P.aeruginosa 103 1 16 0.96 ¹MIC, Minimum Inhibitory Concentration ²FICI,Fractional Inhibitory Concentration Index

TABLE 41 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-15 and Cefepime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-15 1 Cefepime 32 1.29 P. aeruginosa 1384 1 20.79 P. aeruginosa 1474 1 2 1.12 P. aeruginosa 1479 1 4 1.12 P.aeruginosa 2566 0.5 8 1.37 P. aeruginosa 2568 1 2 1.12 P. aeruginosa 1031 1 1.12 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 42 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-8-2 and Cefotaxime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² K. pneumoniae 1346 MB-8-2 0.5 Cefotaxime 0.06 1.37 K. pneumoniae1355 0.5 0.06 1.37 K. pneumoniae 2238 0.5 16 1.37 K. pneumoniae 2541 10.12 1.12 K. pneumoniae 2546 1 0.25 1.29 K. pneumoniae 2549 1 0.06 1.12P. aeruginosa 103 2 16 1.11 ¹MIC, Minimum Inhibitory Concentration²FICI, Fractional Inhibitory Concentration Index

TABLE 43 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-8-2 and Cefepime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-8-2 2 Cefepime 32 1.23 P. aeruginosa 1384 22 0.80 P. aeruginosa 1474 2 2 1.11 P. aeruginosa 1479 2 4 1.23 P.aeruginosa 2566 2 8 1.23 P. aeruginosa 2568 2 2 0.98 P. aeruginosa 103 21 1.11 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 44 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-11 and Cefotaxime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² K. pneumoniae 1346 MB-11 0.5 Cefotaxime 0.06 1.37 K. pneumoniae1355 0.5 0.06 1.87 K. pneumoniae 2238 0.5 8 1.37 K. pneumoniae 2541 0.50.25 0.73 K. pneumoniae 2546 0.5 0.25 1.37 K. pneumoniae 2549 0.5 0.061.37 P. aeruginosa 103 1 16 1.12 ¹MIC, Minimum Inhibitory Concentration²FICI, Fractional Inhibitory Concentration Index

TABLE 45 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-11 and Cefepime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-11 1 Cefepime 32 1.12 P. aeruginosa 1384 1 21.12 P. aeruginosa 1474 0.5 2 1.12 P. aeruginosa 1479 0.5 8 0.87 P.aeruginosa 2566 0.5 16 0.93 P. aeruginosa 2568 0.5 2 0.87 P. aeruginosa103 1 1 0.12 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

TABLE 46 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-2B and Cefotaxime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² K. pneumoniae 1346 MB-2B 4 Cefotaxime 0.06 1.19 K. pneumoniae 13554 0.06 1.19 K. pneumoniae 2238 4 8 1.64 K. pneumoniae 2541 8 0.25 0.64K. pneumoniae 2546 8 0.25 1.16 K. pneumoniae 2549 8 0.12 0.83 P.aeruginosa 103 2 16 1.11 ¹MIC, Minimum Inhibitory Concentration ²FICI,Fractional Inhibitory Concentration Index

TABLE 47 Summary of Minimum Inhibitory Concentration and FractionalInhibitory Concentration Results for MB-2B and Cefepime Compound 1Compound 2 MIC¹ MIC (μg/mL) (μg/mL) Organism¹ Name Alone Name AloneFICI² P. aeruginosa 1381 MB-2B 4 Cefepime 32 1.09 P. aeruginosa 1384 4 20.94 P. aeruginosa 1474 2 2 0.98 P. aeruginosa 1479 2 4 1.11 P.aeruginosa 2566 2 8 1.23 P. aeruginosa 2568 2 2 1.11 P. aeruginosa 103 22 0.61 ¹MIC, Minimum Inhibitory Concentration ²FICI, FractionalInhibitory Concentration Index

Example 11 The Effect of Bismuth Thiols on Infection in a RattusNorvegicus Femur Critical Defect

The current standard of care for open fractures is irrigation,debridement and antibiotics; this is intended to reduce the bacterialload in the wound to the point that infection does not occur. Despitethese treatments, infections still complicate up to 75% of severe combatopen tibia fractures. Interestingly, even though early infections areoften caused by gram negative bacteria, late infections that areimplicated in healing problems and amputation are due to gram positiveinfections, frequently Staphylococci species (Johnson 2007).

One of the reasons that S. aureus are resistant to standard treatment istheir ability to form a biofilm. Bacteria in biofilms are able to resistconcentrations of antimicrobial compounds which would kill similarorganisms in a culture medium (Costerton 1987).

The aim of this study was to determine whether BTs will reduce infectionin a contaminated open fracture model either on their own or withantibiotics. The contaminated rat femur critical defect model is awell-accepted model and was used for the experiments described in thisExample. This model offers a standardized model for comparing variouspossible treatments and their effects on reducing infection and/orimproving healing.

Compounds (CPD) CPD-8-2 (bismuth pyrithione/butanedithiol; Table 1) andCPD-11 (bismuth pyrithione/ethanedithiol; Table 1) are two analogues ofBIS-Bis that have shown potential against Biofilm secreting bacteria invitro, though with a different spectrum of activity than Bis-EDT.

The three BT formulations, Bis-EDT, CPD-11 and CPD-8-2 (see Table 1)demonstrated inhibitory effects on S. aureus strains in vitro when usedwith and without Tobramicin and Vancomycin in a Poly Methyl Methacrylate(PMMA) cement bead vehicle. Three formulations of microparticulate BTswere produced in a clinically useful hydrogel gel form as describedherein. These BTs were tested suspended in a gel at a concentration of 5mg/ml⁻¹ as has been found to be an appropriate concentration for geldelivery. The gel formulations conformed to the wound contours, and didnot require removal following application.

Two treatment arms were used: in the first arm, BT was used singularly;in the second arm BT was used in conjunction with a systemic antibiotic(ABx).

(a) BT Singularly.

Six hours after inoculation with S. aureus, the wound was debrided,irrigated with saline and 1 ml of BT gel inserted within the defect.

(b) BT with Systemic Antibiotics (ABx).

Six hours after inoculation with S. aureus, the wound was debrided,irrigated with saline and 1 ml of BT gel added inserted within thedefect. The antibiotic used was Cefazolin at a dose equivalent to 5mgKg⁻¹ delivered via sub-cutaneous injection twice daily for a total of3-days following the injury. The first dose was administered immediatelyprior to debridement. Previous data suggested that this dose wouldresult in a reduction in bacteria levels from ≈10⁶ to ≈10⁴ and thereforestill allow the relative effect of different BTs to be measured.

(c) Control

Six hours after inoculation with S. aureus, the wound was debrided andirrigated with saline. The control animals were also treated withCefazolin as per the regime described above.

Procedure:

The procedure for the in vivo rat injury model was performed asdescribed by Chen et al. (2002 J. Orthop. Res. 20:142; 2005 J. Orthop.Res. 23:816; 2006 J. Bone Joint Surg. Am. 88:1510; 2007 J. Orthop.Trauma 21:693). The rats were anesthetized and prepped for surgery. Theanterolateral aspect of the femoral shaft was exposed through a 3-cmincision. The periosteum and attached muscle was stripped from the bone.A polyacetyl plate (27×4×4 mm) was placed on the anterolateral surfaceof the femur. The plates were predrilled to accept 0.9-mm diameterthreaded Kirschner wires. The bases of these plates were formed to fitthe contour of the femoral shaft. Pilot holes were drilled through bothcortices of the femur using the plate as a template and threadedKirschner wire was inserted through the plate and femur. The notchesthat were 6 mm apart on the plate served as a guide for bone removal. Asmall oscillating saw was used to create the defect while the tissue wascooled by continuous irrigation in an effort to prevent thermal damage.

Several groups of 10 animals each were inoculated with 1×10⁵ CFU of S.aureus and treated with BT alone or in combination with antibiotics 6hours post-inoculation as described above. The groups were as follows:Bis-EDT gel; MB-11 gel; MB-8-2 gel; Bis-EDT gel & Abx; MB-11 gel & Abx;MB-8-2 gel & Abx; Control (Abx alone).

Animals were euthanized 14 days after surgery and bone and hardware sentfor microbiological analysis, the results of which are shown in FIG. 7.

Based on the power analysis, 10 animals per group will give a power of80% to detect a 25% difference between the treatment and control groups.This is with an expected standard deviation of 35% and alpha of 0.05.

As shown in FIG. 7, in combination with Bis-EDT, MB-11 and MB-8-2,Cefazolin antibiotic activity was enhanced as compared to Cefazolin orany of the Bis compounds alone to reduce S. aureus infection of injuredbone. Cefazolin in combination with MB-11 and MB-8-2 showed enhancedantibiotic activity as compared to Cefazolin alone to reduce S. aureusinfection detected on hardware. Bis-EDT did not appear to affectCefazolin activity in this capacity.

REFERENCES

-   Costerton J W, Cheng K J, Geesey G G, et al. Bacterial Biofilms in    Nature and Disease. Ann Rev Microbiol. 1987; 41:435-64-   Domenico P, Baldassarri L, Schoch P E, Kaehler K, Sasatsu M, Cunha    B A. Activities of Bismuth Thiols against Staphylococci and    Staphyloccocal Biofilms. Antimicrob Agents and Chemother. 2001;    45(5):1417-21-   Halwani M, Blomme S, Suntres Z E, et al. Liposomal    bismuth-ethanedithol formulation enhances antimicrobial activity of    tobramycin. Int J. Pharm. 2008; 358:278-84-   Johnson E N, Burns T C, Hayda R A, Hospenthal D R, Murray C K.    Infection complications of open type III tibial fractures among    combat casualties. Clin Infect Dis. 2007; 45(4):409-415

OTHER CITED DOCUMENTS AND RELATED DOCUMENTS

Domenico et al., Canadian J. Microbiol. 31:472-78 (1985); Domenico etal., Reduction of capsular polysaccharides and potentiation ofaminoglycoside inhibition in gram-negative bacteria with bismuthsubsalicylate. J Antimicrob Chemo 1991; 28:801-810; Domenico et al.,Infection 20:66-72 (1992); Domenico et al., Infect. Immun. 62:4495-99(1994); Domenico et al., J. Antimicrol. Chemother. 38:1031-40 (1996);Domenico et al., Enhancement of bismuth antibacterial activity withlipophilic thiol chelators. Antimicrob Agents Chemother 1997;41:1697-703; Domenico et al., Surface antigen exposure bybismuth-dimercaprol suppression of Klebsiella pneumoniae capsularpolysaccharide. Infect Immun 67:664-669 (1999); Domenico et al., 2000.The potential of bismuth-thiols for treatment and prevention ofinfection. Infect Med 17:123-127; Domenico et al., Activities of bismuththiols against staphylococci and staphylococcal biofilms. AntimicrobAgents Chemother 2001; 45:1417-21; Domenico et al., Combating antibioticresistance with bismuth-thiols. Research Advances in Antimicrob AgentsChemother 2003; 3:79-85; Domenico et al., Reduction of capsularpolysaccharides and potentiation of aminoglycoside inhibition ingram-negative bacteria with bismuth subsalicylate. J Antimicrob Chemo1991; 28:801-810; Domenico et al., BisEDT and RIP act in synergy toprevent graft infections by resistant staphylococci. Peptides 2004.;25:2047-53; Domenico et al., 2005. Pyrithione enhanced antimicrobialactivity of bismuth. Antibiotics for Clinicians 9:291-297; U.S. Pat. No.6,582,719; U.S. RE37,793; U.S. Pat. No. 6,248,371; U.S. Pat. No.6,086,921; U.S. Pat. No. 6,380,248; U.S. Pat. No. 6,582,719; U.S. Pat.No. 6,380,248; U.S. Pat. No. 6,875,453.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

What is claimed is:
 1. A method for protecting a natural surface againstone or more of a bacterial pathogen, a fungal pathogen and a viralpathogen, comprising: contacting the surface with an effective amount ofa BT composition under conditions and for a time sufficient for one ormore of: (i) prevention of infection of the surface by the bacterial,fungal or viral pathogen, (ii) inhibition of cell viability or cellgrowth of substantially all planktonic cells of the bacterial, fungal orviral pathogen, (iii) inhibition of biofilm formation by the bacterial,fungal or viral pathogen, and (iv) inhibition of biofilm viability orbiofilm growth of substantially all biofilm-form cells of the bacterial,fungal or viral pathogen, wherein the BT composition comprises aplurality of solid microparticles that exhibit a unimodal sizedistribution when the BT composition is analyzed on a particle sizeanalyzer and that comprise a bismuth-thiol (BT) compound that has notbeen micronized, milled or subjected to super-critical fluid processing,substantially all of said microparticles having a volumetric meandiameter of from about 0.4 μm to about 5 μm, wherein the BT compoundcomprises bismuth or a bismuth salt and a thiol-containing compound andthe BT composition comprises at least one antibiotic compound that iscapable of acting synergistically with, or enhancing, the BT compound,wherein the antibiotic compound comprises an antibiotic that is selectedfrom methicillin, vancomycin, nafcillin, gentamicin, ampicillin,chloramphenicol, doxycycline, clindamycin, gatifloxacin, cefazolin andan aminoglycoside antibiotic, and wherein the aminoglycoside antibioticis selected from the group consisting of amikacin, arbekacin,gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, and apramycin.
 2. The method of claim 1wherein the bacterial pathogen comprises at least one of: (i) one ormore gram-negative bacteria; (ii) one or more gram-positive bacteria;(iii) one or more antibiotic-sensitive bacteria; (iv) one or moreantibiotic-resistant bacteria; (v) a bacterial pathogen that is selectedfrom the group consisting of Staphylococcus aureus (S. aureus), MRSA(methicillin-resistant S. aureus), Staphylococcus epidermidis, MRSE(methicillin-resistant S. epidermidis), Mycobacterium tuberculosis,Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant P.aeruginosa, Escherichia coli, enterotoxigenic E. coli, enterohemorrhagicE. coli, Klebsiella pneumoniae, Clostridium difficile, Heliobacterpylori, Legionella pneumophila, Enterococcus faecalis,methicillin-susceptible Enterococcus faecalis, Enterobacter cloacae,Salmonella typhimurium, Proteus vulgaris, Yersinia enterocolitica,Vibrio cholera, Shigella flexneri, vancomycin-resistant Enterococcus(VRE), Burkholderia cepacia complex, Francisella tularensis, Bacillusanthracis, Yersinia pestis, Pseudomonas aeruginosa, Streptococcuspneumonia, penicillin-resistant Streptococcus pneumonia, Escherichiacoli, Burkholderia cepacia, Bukholderia multivorans, Mycobacteriumsmegmatis and Acinetobacter baumannii.
 3. The method of claim 1 in whichat least one of: (a) the bacterial pathogen exhibits resistance to anantibiotic that is selected from the group consisting of methicillin,vancomycin, naficilin, gentamicin, ampicillin, chloramphenicol,doxycycline and tobramycin, (b) the surface comprises an epithelialtissue surface that is selected from the group consisting of epidermis,dermis, respiratory tract, gastrointestinal tract and glandular linings,(c) the step of contacting is performed one or a plurality of times, (d)at least one step of contacting comprises one of spraying, irrigating,dipping and painting the surface, (e) at least one step of contactingcomprises one of inhaling, ingesting and orally irrigating, (f) at leastone step of contacting comprises administering to a subject by a routethat is selected from topically, intraperitoneally, orally,parenterally, intravenously, intraarterially, transdermally,sublingually, subcutaneously, intramuscularly, transbuccally,intranasally, via inhalation, intraoccularly, intraauricularly,intraventricularly, subcutaneously, intraadiposally, intraarticularlyand intrathecally, and (g) the BT composition comprises one or more BTcompounds selected from the group consisting of BisBAL, BisEDT,Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr,Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol. 4.A method for protecting a natural surface against one or more of abacterial pathogen, a fungal pathogen and a viral pathogen, comprising:contacting the surface with an effective amount of a BT compositionunder conditions and for a time sufficient for one or more of: (i)prevention of infection of the surface by the bacterial, fungal or viralpathogen, (ii) inhibition of cell viability or cell growth ofsubstantially all planktonic cells of the bacterial, fungal or viralpathogen, (iii) inhibition of biofilm formation by the bacterial, fungalor viral pathogen, and (iv) inhibition of biofilm viability or biofilmgrowth of substantially all biofilm-form cells of the bacterial, fungalor viral pathogen, wherein the BT composition comprises a plurality ofsolid microparticles that exhibit a unimodal size distribution when theBT composition is analyzed on a particle size analyzer and that comprisea bismuth-thiol (BT) compound which comprises bismuth or a bismuth saltand a thiol-containing compound, substantially all of saidmicroparticles having a volumetric mean diameter of from about 0.4 μm toabout 5 μm, wherein at least one of: (a) the synergizing antibiotic orthe cooperative antimicrobial efficacy enhancing antibiotic comprises anantibiotic that is selected from the group consisting of anaminoglycoside antibiotic, a carbapenem antibiotic, a cephalosporinantibiotic, a fluoroquinolone antibiotic, a glycopeptide antibiotic, alincosamide antibiotic, a penicillinase-resistant penicillin antibiotic,and an aminopenicillin antibiotic, and (b) the synergizing antibiotic orthe cooperative antimicrobial efficacy enhancing antibiotic is anaminoglycoside antibiotic that is selected from the group consisting ofamikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin,wherein at least one of: (a) the bacterial pathogen exhibits resistanceto an antibiotic that is selected from the group consisting ofmethicillin, vancomycin, naficilin, gentamicin, ampicillin,chloramphenicol, doxycycline and tobramycin, (b) the surface comprisesan epithelial tissue surface that is selected from the group consistingof epidermis, dermis, respiratory tract, gastrointestinal tract andglandular linings, (c) the step of contacting is performed one or aplurality of times, (d) at least one step of contacting comprises one ofspraying, irrigating, dipping and painting the surface, (e) at least onestep of contacting comprises one of inhaling, ingesting and orallyirrigating, (f) at least one step of contacting comprises administeringto a subject by a route that is selected from topically,intraperitoneally, orally, parenterally, intravenously, intraarterially,transdermally, sublingually, subcutaneously, intramuscularly,transbuccally, intranasally, via inhalation, intraoccularly,intraauricularly, intraventricularly, subcutaneously, intraadiposally,intraarticularly and intrathecally, and (g) the BT composition comprisesone or more BT compounds selected from the group consisting of BisBAL,BisEDT, Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE,Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol, andwherein the method further comprises contacting the surface with atleast one of (i) a synergizing antibiotic and (ii) a cooperativeantimicrobial efficacy enhancing antibiotic, simultaneously orsequentially and in any order with respect to the step of contacting thesurface with the composition wherein at least one of: (A) thesynergizing antibiotic or the cooperative antimicrobial efficacyenhancing antibiotic comprises an antibiotic that is selected from thegroup consisting of an aminoglycoside antibiotic, a carbapenemantibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, aglycopeptide antibiotic, a lincosamide antibiotic, apenicillinase-resistant penicillin antibiotic, and an aminopenicillinantibiotic, and (B) the synergizing antibiotic or the cooperativeantimicrobial efficacy enhancing antibiotic is an aminoglycosideantibiotic that is selected from the group consisting of amikacin,arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin and apramycin.
 5. The methodof claim 4 which comprises overcoming antibiotic resistance where anantibiotic-resistant bacterial pathogen is present on the naturalsurface.
 6. The method of claim 5 in which at least one of: (a) thebacterial pathogen is selected from the group consisting ofStaphylococcus aureus (S. aureus), MRSA (methicillin-resistant S.aureus), Staphylococcus epidermidis, MRSE (methicillin-resistant S.epidermidis), Mycobacterium tuberculosis, Mycobacterium avium,Pseudomonas aeruginosa, drug-resistant P. aeruginosa, Escherichia coli,enterotoxigenic E. coli, enterohemorrhagic E. coli, Klebsiellapneumoniae, Clostridium difficile, Heliobacter pylori, Legionellapneumophila, Enterococcus faecalis, methicillin-susceptible Enterococcusfaecalis, Enterobacter cloacae, Salmonella typhimurium, Proteusvulgaris, Yersinia enterocolitica, Vibrio cholera, Shigella flexneri,vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia complex,Francisella tularensis, Bacillus anthracis, Yersinia pestis, Pseudomonasaeruginosa, Streptococcus pneumonia, penicillin-resistant Streptococcuspneumonia, Escherichia coli, Burkholderia cepacia, Bukholderiamultivorans, Mycobacterium smegmatis and Acinetobacter baumannii, (b)the bacterial pathogen exhibits resistance to an antibiotic that isselected from the group consisting of methicillin, vancomycin,naficilin, gentamicin, ampicillin, chloramphenicol, doxycycline,tobramycin, clindamicin and gatifloxacin, (c) the surface comprises anepithelial surface of a tissue that is selected from the groupconsisting of epidermis, dermis, respiratory tract, gastrointestinaltract and glandular linings, (d) the step of contacting is performed oneor a plurality of times, (e) at least one step of contacting comprisesone of spraying, irrigating, dipping, coating and painting the surface,(f) at least one step of contacting comprises one of inhaling, ingestingand orally irrigating, (g) at least one step of contacting comprisesadministering to a subject by a route that is selected from topically,intraperitoneally, orally, parenterally, intravenously, intraarterially,transdermally, sublingually, subcutaneously, intramuscularly,transbuccally, intranasally, via inhalation, intraoccularly,intraauricularly, intraventricularly, subcutaneously, intraadiposally,intraarticularly and intrathecally, (h) the BT composition comprises oneor more BT compounds selected from the group consisting of BisBAL,BisEDT, Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE,Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT,Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol. 7.An antiseptic composition for treating a natural surface that containsbacterial biofilm, comprising at least one of: (1) a composition thatcomprises (a) at least one BT composition that comprises a plurality ofsolid microparticles that exhibit a unimodal size distribution when thecomposition is analyzed on a particle size analyzer and that comprise abismuth-thiol (BT) compound which comprises bismuth or a bismuth saltand a thiol-containing compound, substantially all of saidmicroparticles having a volumetric mean diameter of from about 0.4 μm toabout 5 μm; and (b) at least one antibiotic compound that is capable ofacting synergistically with, or enhancing, the BT compound, wherein theantibiotic compound comprises an antibiotic that is selected frommethicillin, vancomycin, nafcillin, gentamicin, ampicillin,chloramphenicol, doxycycline, clindamycin, gatifloxacin, cefazolin andan aminoglycoside antibiotic, and (2) the composition of (1) wherein theaminoglycoside antibiotic is selected from the group consisting ofamikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, and apramycin.
 8. Thecomposition of claim 7 wherein the BT compound is selected from thegroup consisting of BisBAL, BisEDT, Bis-dimercaprol, Bis-DTT,Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT,Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT,Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, andBis-EDT/2-hydroxy-1-propanethiol.
 9. A method for treating a naturalsurface that contains bacterial biofilm, comprising: (a) identifying abacterial infection in or on the surface as comprising one of (i) grampositive bacteria, (ii) gram negative bacteria, and (iii) both (i) and(ii); and (b) administering to the surface a formulation that comprisesone or more bismuth thiol (BT) compositions and at least one antibioticcompound that is capable of acting synergistically with, or enhancing,the BT compound, wherein the antibiotic compound comprises an antibioticthat is selected from methicillin, vancomycin, nafcillin, gentamicin,ampicillin, chloramphenicol, clindamycin, gatifloxacin, cefazolin and anaminoglycoside antibiotic, and wherein the aminoglycoside antibiotic isselected from the group consisting of amikacin, arbekacin, gentamicin,kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin,streptomycin, and apramycin, wherein: (i) if the bacterial infectioncomprises gram positive bacteria, then the formulation compriseseffective amounts of at least one BT compound and at least oneantibiotic that is rifamycin, (ii) if the bacterial infection comprisesgram negative bacteria, then the formulation comprises effective amountsof at least one BT compound and amikacin, (iii) if the bacterialinfection comprises both gram positive and gram negative bacteria, thenthe formulation comprises effective amounts of one or a plurality of BTcompounds, rifamycin and amikacin, and thereby treating the surface,wherein the BT compound comprises bismuth or a bismuth salt and athiol-containing compound.
 10. The method of claim 9 wherein thebacterial infection comprises one or a plurality of antibiotic-resistantbacteria.
 11. The method of claim 10 wherein treating comprises at leastone of: (i) eradicating the bacterial biofilm, (ii) reducing thebacterial biofilm, and (iii) impairing growth of the bacterial biofilm.12. The method of claim 9 wherein the BT composition comprises aplurality of solid microparticles that exhibit a unimodal sizedistribution when the composition is analyzed on a particle sizeanalyzer and that comprise a bismuth-thiol (BT) compound, substantiallyall of said microparticles having a volumetric mean diameter of fromabout 0.4 μm to about 5 μm, wherein the BT compound comprises bismuth ora bismuth salt and a thiol-containing compound.
 13. The method of claim12 wherein the BT compound has not been micronized, milled or subjectedto super-critical fluid processing.